Tsetse flies (Diptera: Glossinidae) harbour two morphologically different endosymbionts intracellularly associated with gut tissue: a primary (P) and a secondary (S) organism. The P-endosymbiont is a gram-negative rod, 8-10 microns in size, and resides intracellularly within specialized cells, mycetocytes which are organized into an organelle (mycetome), in the anterior portion of the gut. The S-endosymbiont is a smaller (1-2 microns) gram-negative rod and is harboured in the epithelial sheath cells in midgut. Phylogenetic characterization of S-endosymbionts from taxonomically distant insects including tsetse flies has shown that they are related to the free-living bacterium, Escherichia coli, and are members of the family Enterobacteriaceae within the gamma-3 subdivision of Proteobacteria. In this study, a polymerase chain reaction (PCR) based assay was designed utilizing the conserved sequences of 16S rDNA in order to phylogenetically characterize the mycetome-associated P-endosymbionts directly from tsetse mycetome tissue. Analysis from five species of flies representing the three major subgenera of genus Glossina indicates that P-endosymbionts constitute a distinct lineage within the gamma-3 subdivision of Proteobacteria. Mycetome endosymbiont phylogeny appears to parallel the classic taxonomic assignments independently developed for their insect host species. This suggests an ancient association for this symbiosis, which may have subsequently radiated with time, giving rise to the current species of tsetse flies and their modern-day endosymbionts. Based on endosymbiont phylogeny, the fusca flies constitute the most ancient subgenus, followed by the morsitans and palpalis groups.