The amount of genetic differentiation between the African wild cat (Felis lybica) and the domestic cat (F. catus) in southern Africa is not known, but there is concern that interbreeding between the two may have been sufficiently extensive to threaten the status of the wild cat as a genetically distinct population. Eight hypervariable microsatellite loci were therefore used to determine the genetic relationships within and among wild and domestic cat populations. Measures of genetic differentiation, including FST and RST, confirmed significant differentiation between the wild and domestic cat populations and no significant differentiation between two widely separate domestic cat populations. Our data indicate that the level of genetic introgression is lower than previously thought. This enhances the conservation status of the African wild cat and demonstrates the need for the implementation of feral cat control in and around game reserves and rural areas.