On-loan Derby County defender Farrend Rawson played the full 90 minutes in the encounter at the New York Stadium on April 6 as Rotherham earned a crucial three points in their fight against relegation.
A Football League statement read: "The Football League has charged Rotherham United with fielding an ineligible player [Farrend Rawson] in the club's Sky Bet Championship match with Brighton and Hove Albion on Monday, April 6, which the Millers won 1-0.
"The matter will be heard by a Football Disciplinary Commission which will take place at the earliest possible opportunity to ensure that it reaches its conclusion before the end of the current season."
Rotherham are 21st in the Championship and seven points clear of the relegation zone.
Steve Evans' men may be hit with a points deduction, which would give the likes of Wigan Athletic and Millwall renewed hope in their bid to beat the drop with just four matches remaining.
Earlier this season, Blackpool were fined £30,000 for fielding an ineligible player when Donervon Daniels was selected to play in the 2-1 defeat to Millwall, despite his youth loan having expired.
Last year AFC Wimbledon were docked three points for the same offence in the 4-3 victory over Cheltenham Town.
