The experienced central defender picked up a hamstring injury during Amakhosi's 1-0 win against Bamenda in the Champions League preliminary round first-leg match in Limbe, Cameroon, over the weekend.
Mphahlele has joined Serbian centre forward Samir Nurkovic on the Naturena-based giants' injury list.
Chiefs released the following statement on Tuesday as they revealed that Mphahlele will be assessed ahead of the second-leg encounter:
"Amakhosi return from Cameroon with an injury concern of defender Ramahlwe Mphahlele who was substituted by Yagan Sasman during the win over Bamenda," a club statement read.
"He has a hamstring strain and will be further assessed on Tuesday and Wednesday to ascertain the extent of the injury and his availability for the second leg.
"Rama’s injury aside, Chiefs will be looking to history for some inspiration; in the second leg, since first competing in the Caf Champions League in 1993, Chiefs have always managed to win the home leg of a preliminary round. Doing so in 1993, 2005, 2014, 2015 and 2016."
Mphahlele, who has struggled with injuries in the last few seasons, has made seven appearances across all competitions for the Soweto giants this season.
Amakhosi head coach Gavin Hunt could look to Yagan Sasman once again if Mphahlele fails to recover in time for the crucial match which is scheduled to be played on Friday.
Chiefs are set to play host to Bamenda at FNB Stadium in Johannesburg as the Glamour Boys look to advance to the first round of Africa's most prestigious club competition.
The Soweto giants, who are leading 1-0 on aggregate, will book their place in the first round if they avoid a defeat against the reigning Cameroonian champions.
The winner on aggregate between Chiefs and Bamenda will lock horns with Angolan outfit CD Primeiro de Agosto over two legs in the first round tie.
Primeiro de Agosto were given a preliminary stage bye after they reached last season's Champions League group stage.