Diversities at nuclear and mitochondrial loci were examined in eleven natural populations of Glossina pallidipes from east and southern Africa. Alleles in each class of loci are assumed to be selectively neutral. Allozyme gene diversities (heterozygosities) averaged over eight loci were 0.146 among seven Kenya populations and 0.201 among four southern African populations. Microsatellite diversity averaged over three loci was 0.250 in Kenya and only 0.218 in southern Africa. Mitochondrial diversities averaged 0.504 in Kenya and only 0.156 in southern Africa. Mitochondrial and microsatellite diversities in the populations were strongly correlated with each other, but uncorrelated with allozyme diversities. In contrast to the allozyme diversities, mitochondrial and microsatellite variation indicated a severe and prolonged reduction in population size in southern Africa. Genetic distances among populations increased with the geographical distances between them. Allozyme diversities in southern populations were conserved. Genetic differentiation at allozyme loci among populations was greatly damped when compared with the other markers. The foregoing can be explained if allozyme diversities were maintained by balancing selection. Three main points emerged: genetic data confirm the historical evidence that southern G. pallidipes populations experienced a severe and prolonged bottleneck; allozyme variation was conserved in the bottlenecked populations; and gene flow among populations is surprisingly restricted.