Luton and Portsmouth have been fined £15,000 for failing to field 'full-strength' sides in the EFL Trophy, with 12 clubs in total being punished.
Bradford, Blackpool, Bristol Rovers, MK Dons, Millwall, Charlton, Peterborough, Sheffield United and Southend have been fined £3,000 and Fleetwood £5,000.
Teams had to field five players who started the previous or following game, or five who had made the most appearances this season.
The clubs have the right to appeal.
The English Football League (EFL) say they considered "mitigating factors" and considered "transgressions that were not within the spirit of the rules".
Sides that did not put out 'full-strength' teams were liable to a fine of £5,000 for each group match by the EFL.
League Two Portsmouth and Luton and were found to breach the rule in each of their three matches in the opening round of the competition, with the Hatters' fine coming out of their £20,000 prize money for progressing to the next stage.
Pompey went out of the trophy after finishing third in their group.
'We are staggered'
Luton chief executive Gary Sweet said the club do not feel they should be "paying 'fees' to get our youngsters experience".
"We entered those teams with our eyes wide open and we accept that we would be fined for doing so," Sweet said.
"We are staggered, however, that we have been fined the maximum amount for our first offence, which was winning away from home at a club from the division above with half-a-dozen first-team regulars in their team.
"We played nine graduates of our academy in that game at Gillingham, and seven against a West Brom side containing four players, two of whom who were internationals and had been transferred for several million pounds, and still beat both."
Revamp marred by low attendances
This season's EFL Trophy has been revamped with the introduction of 16 development sides from the Premier League and Championship.
But it has been marred by criticism from managers and fans and low attendances, with Northampton drawing their lowest-ever crowd of 762 for their game against West Ham's under-21 team on 9 November.
Just 284 watched West Brom's academy side play at home to Gillingham - a number, according to the Gills' Twitter account, that included scouts and press - and 308 saw Middlesbrough's development side lose 3-0 at home to Shrewsbury during the final round of group matches.
The 32 clubs who went through, including eight development sides, have been told they must continue to field 'full-strength' teams for the remainder of the competition.
The rules are different for the academy clubs, with six of the starting XI having to be aged under 21.