Orlando Pirates made a good start to the game and were ahead in the 20th minute thanks to a looped free kick from Sifiso Myeni.
However, the two sides had to settle for a penalty shoot-out as the game finished without any further change to the scoreline.
Letsholonyane and Keegan Richie both had their spot-kick efforts saved in the shootout as Tendai Ndoro fired home the winning penalty to seal a 4-3 win for the Buccaneers.
Pirates looked the better side early on and were nearly ahead after eight minutes, Myeni putting Thamsanqa Gabuza through on goal, only to be denied by an alert Chiefs defence.
Gabuza had a similar chance on 17 minutes, though this time he was unable to make the most of the chance with no real conviction behind his shot.
Chiefs stopper Reyaad Pieterse had to be at his best to keep out Thabo Rakhale's curling effort with a fingertip save.
The referee, though, pulled play back for a foul in the build up and Myeni made the most of the second chance, curling his effort from the free-kick over the wall and into the bottom corner.
Chiefs finally mustered a shot of their own with 34 minutes on the clock, however Letsholonyane's effort was well over Brighton Mhlongo's goal.
The Glamour Boys continued to gain momentum in the game and David Zulu nearly had them level as he just volleyed wide at the near post after being picked out by George Lebese.
Chiefs started the second half as they ended the first and really should have been back on level pegging just two minutes after the restart after Bernard Parker picked Siphiwe Tshabala at the far post, though he somehow conspired to head wide from close range.
Tshabala had another great chance to score moments later, though his shot from a tight angle was parried away and then smothered by Mhlongo, with Zulu trying to get on the rebound.
And AmaKhosi finally got the breakthrough they were looking for on 65 minutes, Parker showing a deft touch to put Lesholonyane through on goal, the midfielder showing excellent composure to roll the ball into the back of the net.
The rest of the game passed without any real threat to either goal, with neither side able to make the most of any of their half-chances and the result would be settled by penalties.
Letsholonyane and Keegan Richie both had their efforts saved in the shootout as Pirates saw out the 4-3 win, despite Patrick Phungwayo's miss.