The decision to include 16 invited teams from clubs, largely from the Premier League, with Category One academies, to field a junior side in the competition has divided opinion.
But the EFL have confirmed that a total of 64 clubs, including Notts County and Mansfield Town - but not Nottingham Forest - will compete in the competition beginning with 16 groups of four teams, each of which will include at least one club from each of Leagues One and Two, along with one invited team.
The top two sides in each group will then go into a knockout stage that will culminate in the final at Wembley Stadium on April 2.
The new-look competition will also have an enhanced prize fund with an expected £1.95m being up for grabs, including £10,000 per win and £5,000 per draw during the Group Stage and £100,000 for the eventual winner.
The first 16 clubs with Category One Academies that have been invited to take part in the EFL Trophy 2016/17 are Arsenal, Chelsea, Everton, Leicester City, Liverpool, Manchester City, Manchester United, Middlesbrough, Newcastle United, Southampton, Stoke City, Sunderland, Swansea City, Tottenham Hotspur, West Bromwich Albion and West Ham United.