Bidvest Wits, who beat Orlando Pirates on Sunday to reach this stage, will host Chippa United, while Bloemfontein Celtic are at home to Maritzburg United.
Chiefs and Highlands have already met twice this season in the league, with Ernst Middendorp’s side winning both matches, 3-2 at Highlands and 3-0 at home.
In a true David and Goliath encounter, meanwhile, Vaal University of Technology, who play in the fourth tier of South African football, were rewarded for their last 32 win over Golden Arrows with a trip to face reigning Absa Premiership champions, Telkom Knockout winners and Caf Champions League quarterfinalists Mamelodi Sundowns.
Nedbank Cup last 16 draw
Polokwane City/Baroka FC v Hungry Lions
Zizwe United/Happy Wanderers v TS Sporting
Mbombela United v Real Kings
Mamelodi Sundowns v Vaal University
Bidvest Wits v Chippa United
Highlands Park v Kaizer Chiefs
Bloemfontein Celtic v Maritzburg United
Black Leopards v Amavarara