Species- and strain-specific DNA probes were used to identify patent midgut infections in Glossina morsitans submorsitans and G. palpalis gambiensis captured at four sites in The Gambia. 52% of mature Nannomonas infections and 12% of immature infections were identified. Trypanosoma (Nannomonas) simiae accounted for the majority of identified infections in G.m. submorsitans, indicating the importance of distinguishing this species from the closely related T.(N) congolense when assessing the trypanosomiasis challenge to cattle. Both the savannah and riverine-forest groups of T. congolense were present, although the riverine-forest form was found only in G.p. gambiensis at Pirang, an isolated area of forest. Two-thirds of the samples remain unidentified by probes specific for: Trypanozoon; T. congolense savannah, riverine-forest and Kenya coast forms; T. simiae; and T. vivax, probably owing in part to low numbers of trypanosomes. However, the failure to identify several heavy Nannomonas infections, strongly suggests the presence of a further, as yet unknown, kind of Nannomonas.