Transboundary protected areas may be important for the conservation of large African carnivores because these species require large tracts of habitat to maintain viable population numbers and gene flow. Cheetah (Acinonyx jubatus Schreber, 1775), is a species that may benefit from transboundary conservation agreements. It occurs at naturally low population densities, makes use of large home ranges, and disperses over long distances, thus requiring large tracts of suitable habitat to maintain viable population numbers. Here we present the first scientific evidence of a breeding population of cheetah in Parque Nacional do Limpopo (PNL), Mozambique. We obtained data from camera-traps deployed during occupancy surveys conducted from 9 September 2011 to 31 August 2012 over a 3400 km² study area located within the central third of PNL. These results highlight the importance of the Greater Limpopo Transfrontier Park (GLTFP) to the overall conservation of cheetah in Africa, and the potential value of transboundary protected areas for the conservation of wide-roaming terrestrial mammals.