Glossina pallidipes, a widely but discontinuously distributed African savanna species, is one of the economically important tsetse flies because it is a vector of trypanosomiasis, a lethal disease of cattle and other domestic animals. DNA sequences of ribosomal (r16S2, 249 bp) and cytochrome oxidase I (COI, 421 bp) concatenated mitochondrial genes were analysed in 23 geographically diverse samples of G. pallidipes from Ethiopia, Kenya, Tanzania, Zambia and Zimbabwe. Among 873 flies, we detected 181 composite haplotypes and found that their spatial diversities and frequency distributions were heterogeneous. Haplotype and nucleotide diversities were greatest in Ethiopia and least in southern Africa. We observed little haplotype and nucleotide diversity among regions, and detected severely limited maternal gene flow among the sampled populations (ΦST= 0.42). Tests for demographic stability and analysis of mismatch distributions revealed regionally contrasting demographic histories. The Ethiopian populations were phylogenetically the oldest and genetically the most diverse, and exhibited successive waves of contraction and expansion. The southern African populations were phylogenetically the youngest and genetically the least diverse, and showed only a single, recent expansion. Likely ecological correlates of historical tsetse fly demography include population suppression trials in East Africa and recurring rinderpest epizootics in southern Africa, beginning in the late nineteenth century that reduced host mammalian populations.