The player in question is central defender Farrend Rawson who played the full 90 minutes as the Millers won the match at the New York Stadium 1-0.
Rawson, 18, was on loan at Rotherham from Derby and was making the fourth appearance of his loan spell after joining them on a 28-day youth loan on March 7, which would have expired on April 4.
Rotherham boss Steve Evans claimed after the Brighton game that Rawson’s youth loan had been extended until the end of the season.
But Rawson was not involved in Rotherham’s 2-0 defeat at Middlesbrough on Saturday, with Evans adding to the confusion afterwards by saying he had been recalled by the Rams.
The matter will now 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 currently in 21st place in the Championship table and seven points clear of the relegation zone, but could face a points deduction if they are found guilty – AFC Wimbledon were deducted three points for the same offence last season in a 4-3 victory over Cheltenham.
After being charged, the club issued a statement which read: “Rotherham United FC have received a charge in relation to fielding an unregistered player.
“The case will be heard before the FDC and 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.”