Boro's U23s have been drawn against Scunthorpe United, Cambridge United and Shrewsbury Town in this season's EFL Trophy.
In a first for the competition, which has been running since 1983, Category 1 Academy teams have been invited to participate, alongside clubs from League 1 and League 2.
The 64-team competition will begin with the regionalised four-team group stage commencing at the end of August.
Boro's U23s will face each Northern Group G opponent once, either home or away.
The top two sides in each group will proceed to a straight knockout (one leg) competition, with the final to be played at Wembley Stadium on April 2 next year.
Match Day 1 - w/c 29th August 2016
Scunthorpe United v Middlesbrough
Match Day 2 - w/c 3rd October 2016
Cambridge United v Middlesbrough
Match Day 3 - w/c 7th November 2016
Middlesbrough v Shrewsbury Town