Glossina palpalis gambiensis Vanderplank (Diptera: Glossinidae) from West Africa (Senegal and Burkina Faso) were analysed for microsatellite DNA polymorphisms and size of the wings. In the overall sample a strong heterozygote deficiency was found at two polymorphic microsatellite loci. It led to a highly significant value of Fis (within-sample heterozygote deficit) in the western zone of Sideradougou area in Burkina Faso. Genetic differentiation was significant on a macrogeographic scale, i.e. between tsetse coming from Senegal and Burkina Faso. Wing measures also differed between these two countries; flies from Senegal appeared to be smaller. Microsatellite loci further allowed differentiation of populations of G. palpalis gambiensis trapped on the same hydrographic network a few kilometres apart. The results are interpreted as indicating that further investigations will allow the study of genetic variability of tsetse flies in relation to the dynamics of transmission of human and animal trypanosomoses.