Estimation of allelic frequencies at three microsatellite loci among 20 populations of Glossina morsitans morsitans Westwood, Glossina morsitans submorsitans Newstead, and Glossina morsitans centralis Machado indicated only two of 99 alleles were shared between three subspecies and 18 between any two subspecies; 81 alleles were unshared. The conserved flanking regions of each locus were completely shared. Genetic differentiation among subspecies, based on allele size, was R_ST = 0.87, close to the theoretic maximum value. All evidence suggests longstanding and complete reproductive isolation in nature among the sibling species. They should be elevated to specific rank.