Gas liquid chromatography (GC) was used to analyse the cuticular alkanes of 26 species and subspecies of tsetse flies (Glossina spp.). Unique interspecific and intraspecific (males vs. females) chromatographic patterns were observed. Solvent extraction of dried museum specimens and fresh specimens were equally successful, and left specimens undamaged. GC peaks were used as characters with one of five character states per peak, to show phenetic relationships among species by sex using a UPGMA algorithm, using 23 peaks for males and 40 peaks for females. Comparisons among species of the morsitans group often agreed with recognized morsitans group classification using morphological techniques, with G. austeni Newstead being somewhat aberrant. Glossina tachinoides Westwood was less closely related to others in the palpalis group than previously described using morphology. Eleven members of the rarely studied fusca group were also classified by this methodology.