Manager Paul Cook confirmed after the game that captain Michael Doyle and defender Christian Burgess were involved in an altercation.
Cook decided it was serious enough to immediately substitute both players.
The club's investigation will decide if any further disciplinary action will take place against the players.
"At the end of the game, I was made aware by the manager of an incident which occurred at half-time," chief executive Mark Catlin said.
"As part of the investigation I will again speak to the manager and, if as a result of that, I need to talk to the players, then of course I will."
Portsmouth will conduct an internal investigation after "a dressing room incident" during Saturday's 2-1 League Two home defeat by Stevenage.