Senong yesterday named Mahlambi in his 21-man squad for the CAF event, taking place in Zambia from February 26 to March 12.
However, Wits boss Jose Ferreira is quoted as telling Sowetan Live: "The competition does not fall under the FIFA calender, so we cannot release the player."
The Students, who previously opted against releasing Mahlambi for Amajita duty, are two points behind log leaders Kaizer Chiefs with three games in hand midway through the campaign.
Senong's side have been paired with Senegal, Cameroon and Sudan in Group B of the U-20 AFCON, while Group A comprises Zambia, Egypt, Mali and Guinea.
The top four finishers at the tournament will qualify for the FIFA U-20 World Cup, scheduled for South Korea from May 20 to June 11.