Genetic variation was measured in 105 African buffalo from four populations in South Africa to investigate the effects of habitat fragmentation. Levels of heterozygosity, allelic diversity and genetic differentiation among populations were quantified using seven polymorphic microsatellite markers. There was a significant correlation between the amount of genetic variation and population size, and differentiation was detected among all populations measured by FST and RST. We used likelihood analysis to infer the effective population sizes of each population and to determine whether the fragmented populations were historically differentiated from one another. The genetic estimates of census size were consistent with historical records, and no historical genetic differentiation could be inferred in the original population before fragmentation. These results are discussed in the light of conservation management of fragmented buffalo populations, particularly where natural gene flow is no longer possible.