Lee Bowyer and Lee Williamson must serve three-match suspensions after unsuccessful appeals by Birmingham and Preston respectively.
Birmingham midfielder Bowyer was involved in a scuffle with Williamson during Saturday's 2-1 win for Preston which resulted in both men being dismissed.
Blues had hoped the decision to send off Bowyer would be overturned after submitting written and DVD evidence to the Football Association.
However, an FA regulatory commission has now upheld the original judgement and he will miss the crucial final game of the season against Reading.
"We are disappointed with the decision but are grateful to the FA for hearing the appeal and now have to get on with things despite losing the services of a key member of our squad," Birmingham boss Alex McLeish told the club's official website.
Blues need to beat Reading to guarantee automatic promotion and failure to do so could mean they go into the play-offs.
Preston are just outside the play-off spots and must defeat Queens Park Rangers to stand any chance of finishing in the top six.
Williamson will sit out the home clash and would also have to miss both legs of a possible play-off semi-final.