Using a standard protocol, we conducted vertebrate roadkill surveys in the Greater Mapungubwe Transfrontier Conservation Area (GMTFCA), South Africa, which is a World Heritage Site. A total of 991 roadkill were recorded on the paved roads and 36 roadkill on the unpaved roads. Identifiable roadkill comprised 162 species from 24 orders and 65 families. Ninety-three roadkill could not be identified to species level. Roadkill counts were strongly influenced by road type and season. More roadkill was recorded on the paved than the unpaved roads. Irrespective of road type, the proportion of roadkill was greatest in the hot/wet season (4.3 paved roadkill/km/day paved and 1.3 roadkill/km/day unpaved) and lowest in the cold/dry season (2.0 roadkill/km/day paved and 0.1 roadkill/km/day unpaved). The high numbers of vertebrates identified as roadkill suggests that road traffic has the potential to directly and negatively affect biodiversity conservation in this part of South Africa. We recommend continued roadkill data collection across South Africa to assist with creating an inventory of species most likely to be at risk from roads. This will, in turn, better inform the implementation of potential mitigation measures.