Does the order of missions matter?

Solution 1:

Yes, the mission order does matter

However, most of the missions where the order does matter can be ignored if you're willing to skip portions of the content available in the game. In this instance, you can just play the main story line missions, which pretty much present themselves to you in the order that they can be played in.

For a full completion though, there are a few missions that must be completed in a specific order as they're only available before or after certain events occur in the main story. These are all detailed below.

Notable missions that need completing in order

You need to do the missions in the following order. Failing to complete the missions in an order close to this will result in a variety of missions becoming unavailable, which can impact your war assets and get people killed.

These three missions will occur automatically and do not need any input from you to select a mission;

Prologue: Earth
Priority: Mars
Priority: The Citadel part 1

Once you're off the Citadel for the first time, Priority: Palavan is the only mission available to you;

Priority: Palaven

Once you've rescued the Primark from Palavan, the Grisson Academy mission appears. One of your old team mates (presuming she didn't die in Mass Effect 2), Jack, is present on this station. This mission will turn into Grissom Academy: Emergency Evacuation.

Grissom Academy: Investigation

The next mission involves the rescue of a Krogan female from Sur'Kesh, this will unlock a variety of missions on Tuchanka, one of which is timed (Tuchanka: Bomb) and needs to be completed within three missions, else you will fail it.

Priority: Sur'Kesh
Tuchanka: Turian Platoon
Tuchanka: BOMB
Attican Traverse: Krogan Team

Following the completion of Priority: Sur'Kesh, but BEFORE undertaking Priorty: Tuchanka, the following missions must be completed. Each of these missions will be considered failed if you undertake the Priority: Tuchanka mission without completing these first;

Benning: Evidence
Citadel: Alien Medi-Gel Formula
Citadel: Barla Von
Citadel: Biotic Amp Interfaces
Citadel: Hanar Diplomat
Citadel: Improved Power Grid
Ismar Frontier: Prototype Components

Priority: Tuchanka is a major cut off point, completion of this mission will unlock Priority: The Citadel (part 2), which will result in the Citadel getting attacked by Cerberus. A lot of people that were available to speak to beforehand are either dead, missing, or elsewhere after the completion of this mission. Ensure that all side missions you wanted to undertake that are not included in the "any time" list at the end have been completed before this point.

Priority: Tuchanka
Priority: The Citadel part 2
Citadel: Cerberus Automated Schematics
Lesuss: Ardat-Yakshi Monastery (contains item for Citadel: Asari Widow)
Arrae: Ex-Cerberus Scientist (contains item for Citadel: Cerberus Turian Poison)

The mission Priority: Perseus Veil unlocks the following missions;

Priority: Perseus Veil
Priority: Geth Dreadnought
Rannoch: Admiral Koris (contains item for Citadel: Target Jamming Technology)
Rannoch: Geth Fighter Base (contains item for Citadel: Reaper Code Fragments)
Citadel: Kakliosaur Fossil

The two Rannoch missions (Rannoch: Admiral Koris and Rannoch: Geth Fighter Base) must be completed before undertaking Priority: Rannoch or they will become unavailable.

Priority: Rannoch
Priority: The Citadel part 3
Priority: Thessia
Priority: Horizon

Missions with no time limit

These missions can be completed at any point during the game

Note that while scanning the planet and collecting the items will not count as a mission, landing on the Citadel to hand in the item(s) WILL count as a mission.

It is worth keeping in mind that if you have timed missions up on the galaxy map (Tuchanka: Bomb) that undertaking many of the missions below will count towards the three missions that will result in the expiration of these time sensitive missions.

I personally would recommend completing all of the other missions before undertaking the below.

Collection missions

These missions enable you to maximise your war assets and can be completed at any point in the story. They generally involve collecting an item by scanning a planet and then delivering the item back to an individual on the Citadel.

Apien Crest: Banner of the First Regiment
Athena Nebula: Hesperia-Period Statue
Citadel: CX-12 Thermal Pipe
Citadel: Kakliosaur Fossil
Dekuuna: Code of the Ancients
Dekuuna: Elcor Extraction
Hades Nexus: Obelisk of Karza
Hades Nexus: Prothean Sphere
Irune: Book of Plenix
Kites Nest: Pillars of Strength
Nimbus Cluster: Liberty of Asha
Silean Nebula: Rings of Alune
Shrike Abyssal: Prothean Obelisk
Valhallan Threshold: Prothean Data Drives

Hand ins

Like the collection missions above, except these involve collecting an item from another mission (in brackets) rather than scanning a planet for it. If you complete the mission without picking up the item, some of these items can be purchased via Spectre requisitions.

Citadel: Asari Widow (Lesuss: Ardat-Yakshi Monastery)
Citadel: Cerberus Ciphers (N7: Communications Hub)
Citadel: Cerberus Turian Poison (Arrae: Ex-Cerberus Scientist)
Citadel: Chemical Treatment (N7: Fuel Reactors)
Citadel: Reaper Code Fragments (Rannoch: Geth Fighter Base)
Citadel: Target Jamming Technology (Rannoch: Admiral Koris)

Other missions

These missions generally involve running around from place to place delivering messages and perform odd jobs.

Aria: Blood Pack
Aria: Blue Suns
Aria: Eclipse
Citadel: Batarian Codes
Citadel: Cerberus Retribution
Citadel: Inspirational Stories
Citadel: Medi-Gel Sabotage
Citadel: Medical Supplies
Citadel: Volus Ambassador
Citadel: Wounded Batarian

N7 missions

These missions unlock throughout the game and do not have a time limit or expiration. They're also not tied to any of the important planets/settlements/stations, so can be completed at any point during the game;

N7: Cerberus Abductions
N7: Cerberus Attack
N7: Cerberus Fighter Base
N7: Cerberus Lab
N7: Communications Hub (contains item for Citadel: Cerberus Ciphers)
N7: Fuel Reactors (contains item for Citadel: Chemical Treatment)

Downloadable Content missions

Which missions and when to do them

The DLC content unlocks after the completion of Priority: Palaven (with the exception of From Ashes, which unlocks after Priority: Citadel, part 1) and is not time sensitive content. However, it is worth keeping in mind that if you have timed missions up on the galaxy map, for example Tuchanka: Bomb or Grissom Academy that undertaking DLC missions will count towards the three missions that will result in the expiration of these time sensitive missions.

From Ashes

If you have the From Ashes DLC, you will gain these bonus missions. Priority: Eden Prime will unlock Javik, the Prothean squad member. Eden Prime: Resistance Movement unlocks additional war assets;

Priority: Eden Prime
Eden Prime: Resistance Movement (during mission Priority: Eden Prime)

This mission can be done any time, but getting Javik early on provides some benefits like a funny squad member that details a lot of the background lore and the particle beam rifle. I would recommend doing From Ashes after the completion of Priority: Palaven and before undertaking Priorty: Sur'Kesh.

Omega

The completion of these three missions is required in order to unlock the DLC mission for the Omega DLC;

Aria: Blood Pack
Aria: Blue Suns
Aria: Eclipse

After completing these three missions you will be contacted by Aria T'Loak for the following mission;

Citadel: Aria T'Loak

This mission can be completed any time and will not have an impact on the rest of the game other than some additional war assets.

Leviathan

If you have the DLC pack Leviathan, the following mission will become available to you after the completion of Priority: Palavan;

Citadel: Dr. Bryson

This mission can be completed any time and will not have an impact on the rest of the game other than some additional war assets. It does, however, contain a significant amount of lore and back story.

Citadel

If you have the Citadel DLC, the following mission will become available after the completion of Priority: The Citadel II;

Citadel: Shore Leave

This mission can be completed any time and will not have an impact on the rest of the game.

After everything else: The final missions

Starting these missions signifies the beginning of the end, the only way back after starting Priority: Cerberus headquarters is to either complete both missions and finish the game or load an old save from before taking the mission.

Priority: Cerberus headquarters
Priority: Earth

Once completing these missions, you will be returned to the galaxy map in a "pre mission" state - IE; as if you hadn't started Priority: Cerberus headquarters. This will allow you to re-use that save to complete any missions you didn't complete (that didn't expire). To complete expired missions, you need to start the game again from the beginning (or load a save pre-dating the mission expiring).

Solution 2:

Yes, Priority missions have a chance of making other missions inaccessible, so it's better to complete as many sidequests as possible before proceeding with Priority missions. Off the top of my head, the second Priority: Citadel prevents you from completing Citadel: Barla Von and the Priority: Rannoch prevents you from completing the other 2 Rannoch missions.