The EFL have written to Huddersfield to "request their observations regarding team selection" following their 2-0 defeat to Birmingham City on Saturday.
Huddersfield made 10 changes to the starting line up for their trip to St Andrew's having already confirmed a place in the Championship play-offs, but the EFL said the result would stand.
Blackburn manager Tony Mowbray later questioned Huddersfield's line-up. Blackburn are currently in the final relegation place and two points behind Birmingham City 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."