Sub-Saharan Africa harbours an outstanding diversity of tortoises of which the leopard tortoise Stigmochelys pardalis is the most widespread. Across its’ range the species is impacted by habitat transformation, over-collection for human consumption and the pet trade, road mortality, and electrocution by electric fences. Most leopard tortoises in southern Africa are nowadays restricted to reserves and private farms. So far confiscated tortoises are frequently released into a nearby reserve without knowledge on their area of origin. This is problematic, as it has been demonstrated that the leopard tortoise harbours five distinct mitochondrial lineages, of which three occur in the southern portion of the species’ distri-butional range (South Africa, Namibia, and Botswana). Using 14 microsatellite loci corresponding to 270 samples collect-ed throughout southern Africa, we found a clear substructuring in the north constituting four clusters (western, central, north-eastern, and eastern). Genetic diversity was particularly high in the north-east and decreased towards the south. In addition, we found a significant size difference between the studied populations. Our basic morphological analysis showed that tortoises from the southern cluster tend to grow bigger than tortoises from the north. We established a comprehensive genetic database for South Africa and Namibia that can serve as a conservation management tool for the assignment and potential release of translocated or seized leopard tortoises based on genetic affiliation.