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The midfielder was stretchered off on Wednesday in a practice-training match against Zambia at the Rand Stadium.
He went for an MRI scan on Friday which confirmed the knee injury.
Letsholonyane was the last senior player left in the Bafana Bafana squad after the departure of Morgan Gould and Siboniso Gaxa after the friendly international against Equatorial Guinea.
Gould was captain in Equatorial Guinea while Letsholonyane captained Bafana Bafana against Zambia.
"He was a true soldier, he helped us a lot as he was the only senior player left in the team. He led from the front and showed real leadership.
We will miss his role in the team, and his leadership against the Black Stars of Ghana. It's unfortunate that these things happen in football and we can only wish him a speedy recovery," said Bafana Bafana coach Pitso Mosimane.
Bafana Bafana team doctor, Carl Tabane has already contacted Kaizer Chiefs.
Ï explained to them the results of the scan and the extent of the injury. He is going to see an orthopedic surgeon next week to map the way forward," said Tabane.
Bloemfontein Celtic captain Thabo Nthete will take over the armband against Ghana.
Defender Nazeer Allie will also not be available for Sunday's practice-training match due to a hamstring injury he sustained on Wednesday.
Midfielder Granwald Scott will be assessed after Friday's training session. Scott missed the match against Zambia because of a knee injury.
Bafana Bafana will hold their last training session on Saturday at the Royal Bafokeng Sports Palace in Phokeng near Rustenburg.
