Coach Gordon Igesund said a lack of game time at his Greek club PAOK Salonika meant Khumalo, the former Tottenham Hotspur central defender, would not be sharp enough for the Group A match against the Central African Republic in Cape Town on March 23.
"I chose not to pick him for this game on the strength that he has not been playing for his club," Igesund told a media conference in Johannesburg.
Khumalo was widely criticised for a patchy defensive performance at the tournament South Africa hosted at the start of the year but exited at the quarter-final stage.
Igesund recalled Daylon Claasen of Belgian club Lierse and Israeli-based striker Dino Ndlovu, who missed the Nations Cup because of injury.
South Africa have two points from their first two matches in the group phase of the African qualifiers.
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