U's face Charlton, Crawley and Southampton.
Colchester United will play Southampton, Crawley Town and Charlton Athletic in the EFL Trophy group stages.
The U's were teamed up with their three opponents in Wednesday's draw for the new look competition, which trials in the 2016/17 season.
The matches against Southampton's Academy side and Charlton Athletic will be played at the Weston Homes Community Stadium, whilst we will travel to face Crawley Town.
The dates of the games are as follows:-
Southern Group E
Match Day 1 – w/c 29th August 2016
Charlton Athletic v Southampton
Crawley Town v Colchester United
Match Day 2 – w/c 3rd October 2016
Colchester United v Southampton
Charlton Athletic v Crawley Town
Match Day 3 – w/c 7th November 2016
Colchester United v Charlton Athletic
Southampton v Crawley Town
Details of confirmed dates and ticket prices will be confirmed in due course.