In sub-Saharan Africa, tsetse flies are the vectors of trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals. Certain wild populations of the palpalis group exhibit intraspecific variation and are suspect of manifest differences in vectorial capacity. The current study reports the identification of 13 polymorphic microsatellite loci from Glossina palpalis palpalis Robinean-Desvoidy. The majority of these markers amplify corresponding loci from the related species C. p. gambiensis Vanderplank, G. f. fuscipes Newstead, and G. tachinoides Westwood. Only seven of 13 loci were amplified from G. austeni Newstead. Genetic variability was estimated in one field population of G. p. gambiensis. These results confirmed that microsatellite markers may be used to examine the subpopulation structure of tsetse flies.