The African buffalo (Syncerus caffer) exhibits extreme morphological variability at the continental scale. Today, four subspecies are recognized based on morphological characteristics. Based on the mtDNA D-Loop region and on more than 42,000 SNP genetic markers, the present study aimed to investigate the evolutionary history of the species by inferring the pan-African spatial distribution of its genetic diversity. All analyses converged to the existence of two distinct lineages, corresponding to a group encompassing West and Central African populations and a group encompassing East and Southern African populations. The former is currently assigned to two to three subspecies (S.c. nanus, S.c. brachyceros, S.c. aequinoctialis) and the latter to a separate subspecies (S.c. caffer). 42% of the total amount of genetic diversity is explained by the between-lineage component, with one to seventeen female migrants per generation inferred with the isolation-with-migration model. The divergence time between the two main groups was estimated to have occurred during the late to middle Pleistocene, followed by a population expansion in both lineages. They later adapted morphologically to colonize new habitats, hence developing the variety of ecophenotypes observed today. At the regional scale, 8 populations distributed within these two lineages were identified, resulting from more recent fragmentation processes. This division in two main genetic lineages is also observed on almost all large savanna species with a large distribution pattern, like the waterbuck (Kobus ellipsiprymnus), the hartebeest (Alcelaphus buselaphus), or the roan (Hippotragus equinus). This can be interpreted as common evolutionary responses to past environmental changes.