Morgan Shivambu opened the scoring for Leopards in the 20th minute before Willard Katsande equalised with 12 minutes remaining. But a late strike from Roggert Nyundu secured a historic result for Lidoda Duvha.
The Glamour Boys were minus suspended defenders Tsepo Masilela and Siboniso Gaxa, while striker Bernard Parker missed out after sustaining a knock against Moroka Swallows.
Reyaad Pieterse started in goal ahead of Itumeleng Khune and Brilliant Khuzwayo, with the latter providing cover on the bench. Simphiwe Mtsweni, Ovidy Karuru and new signing David Zulu were also in the mix for the Absa Premiership leaders.
Chiefs dominated the first half even after conceding without creating too many clear-cut chances until Mandla Masango had a strong penalty appeal turned down by referee Daniel Bennett just before half-time.
Amakhosi coach Stuart Baxter was incensed with the decision – especially after Leopards' goal appeared to be from an offside position – and he was sent to the stands for his protestations, putting Chiefs on the back foot.
In the second half Chiefs continued to press for a goal but had to wait until the 78th minute for Katsande to head home substitute Siphiwe Tshabalala's corner-kick.
But Leopards, who came close to upsetting Chiefs in last year's Nedbank Cup as well, were not to be denied as Nyundu's deflected effort sailed past Pieterse in the 90th-minute to win it for Lidoda Duvha.
Chiefs XI: Pieterse, Moleko, Gould, Bukenya, Mtsweni, Katsande, Maluleka, Karuru (Lebese 57'), Masango, Zulu (Tshabalala 59'), Rusike (Mthembu 75').
Subs: Khuzwayo, Mathoho, Tshabalala, Ekstein, Lebese, Mthembu, Nkosi.
Leopards XI: Tshinuna, Nyirenda, Shivambu, Radebe, Mugananga, Maphangule, Ndou, Mabotja, Abubakar, Nyundu, Ntshangase