The Millers, fourth bottom in the Championship and currently seven points clear of both Wigan Athletic and Millwall, must appear before a three-man independent disciplinary panel after being charged by the governing body.
Farrend Rawson, on loan from Derby County, is at the centre of the charge.
The suggestion is that he was not properly registered for the Easter Monday victory by the Millers over Brighton & Hove Albion.
The 18-year-old played the entire 90 minutes, but it is alleged that his youth loan from the Rams had not been extended beyond the already-agreed 28 days. A League statement has promised a decision would be made before the end of the season.
The possible punishments open to the disciplinary panel if Rotherham are found guilty include a warning, a fine or a points deduction.
Precedent suggests the three man panel – made up of a chair who is either a solicitor, barrister or arbitrator, plus a representative nominated by both the Millers and the League – will take into account the result of the game.
For instance, when Blackpool were found guilty in November of fielding defender Donervon Daniels despite his youth loan having expired, the Seasiders – who lost the game 2-1 to Millwall – were fined £30,000.
Last season, however, AFC Wimbledon were docked three points for fielding an ineligible player, Jake Nicholson, in a home game against Cheltenham Town that the Dons won 4-3.
Should an identical punishment be meted out in this case, Rotherham’s advantage over both Wigan and Millwall would be cut to just four points. The second-bottom Lions also have a game in hand on both Steve Evans’s men and the Latics.
A League statement said: “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.”
In acknowledging the charge, a statement from the Millers added: “Such proceedings, under League rules, are confidential until the case is concluded. Therefore we have been advised by our counsel, Jim Sturman QC, to make no further comment until the case is concluded.”