The Terriers made 10 changes for the trip to St Andrew's having sealed a Championship play-off place, but the EFL said the result would stand.
Blackburn boss Tony Mowbray later questioned Town's line-up.
Rovers, who occupy the final relegation place, are two points behind Harry Redknapp's Blues with one game to play.
The EFL said in a statement: "We have today written to Huddersfield Town to request their observations in relation to team selection during their recent Championship match with Birmingham City and, as per our regulations, the EFL executive will refer the matter to the board if it is deemed appropriate to do so.
"It should be noted, however, that the result of Saturday's game will stand in all circumstances and any potential action would be taken against Huddersfield Town directly."
Blackpool and Wolves were fined for fielding much-changed teams in Premier League matches in 2010 and 2009 respectively.
Earlier this season 12 League One and Two teams were fined for playing weakened sides in the Checkatrade Trophy, although that competition had specific regulations regarding team selection.
The EFL has written to Huddersfield to "request their observations regarding team selection" for Saturday's 2-0 defeat by Birmingham City.