Variation at mitochondrial and microsatellite loci was used to study the breeding and dispersal structure of Glossina morsitans centralis, in six natural populations from Botswana, the Caprivi Strip (Namibia), Zambia, and in a laboratory culture derived from Singida, Tanzania. Only seven mitochondrial haplotypes were found. Mean diversity averaged over the six natural populations was 0.216 ± 0.085. The fixation index FST = 0.866 indicated a high degree of genetic differentiation among populations. Fifty-three alleles were detected among six microsatellite loci and six natural populations. Mean microsatellite diversity was 0.702 ± 0.091. Depending on the estimating model used, fixation indices varied from 0.15 to 0.225 confirming that G. m. centralis populations are strongly subdivided. For all FST estimates, positive correlations were detected between pair-wise genetic distance measures and geographical distances. The difference in fixation indices estimated from mitochondrial or nuclear loci was explained by the greater sensitivity of mitochondrial genomes to genetic drift. Population differentiation can be explained by genetic drift and the subsequent recovery of extant populations from small, discontinuous populations. These data confirm genetically the collapse and retreat of G. m. centralis populations caused by the rinderpest epizootic of the late 19th and early 20th centuries.