The ecological functioning of an environment is influenced by how lions, Panthera leo, utilize their prey at various spatial and temporal scales. Aspects of the spatial and temporal variation in lion diet were investigated in the southwestern Kgalagadi Transfrontier Park (KTP) between May 2013 and June 2015 using evidence of prey remains identified through GPS cluster analysis (n = 278) and lion scat analysis (n = 189). Spatial variation in diet was investigated between three regional landscape types (north, south and west), based on the different vegetation types represented within each area, and two terrain types (riverbeds and dunes). Seasonal variations were assessed according to the hot-wet season (November to April), and cold-dry season (May to October). Lion diet profiles varied between landscape types and terrain type, but not seasonally. The most notable spatial differences in lion diet profiles were between the dune and riverbed terrains. Blue wildebeest, Connochaetes taurinus, contributed significantly to lion diet in the riverbeds (36%), but not in the dunes (2%). Gemsbok, Oryx gazella, was the primary contributor to lion intake biomass across all seasons, landscape types, as well as in the dune terrain (range 55–82%). Being largely independent of surface water and the most widespread and abundant of the large prey species, gemsbok are expected to remain the primary food source for lions and are likely to be a key driver of lion population dynamics in the KTP.