Display omitted ► This study reveals multiple-genotypes of T. brucei in the mid-guts of wild tsetse. ► It highlights a wide range of T. brucei genotypes circulating in tsetse mid-guts. ► Multiple genotypes involving T. b. gambiense are 2.2 time the single infections. ► Therefore, multiple infections are frequent in the mid-guts of wild tsetse flies. ► They involve various T. b. gambiense strains and different trypanosome species. To identify Trypanosoma brucei genotypes which are potentially transmitted in a sleeping sickness focus, microsatellite markers were used to characterize T. brucei found in the mid-guts of wild tsetse flies of the Fontem sleeping sickness focus in Cameroon. For this study, two entomological surveys were performed during which 2685 tsetse flies were collected and 1596 (59.2%) were dissected. Microscopic examination revealed 1.19% (19/1596) mid-gut infections with trypanosomes; the PCR method identified 4.7% (75/1596) infections with T. brucei in the mid-guts. Of these 75 trypanosomes identified in the mid-guts, Trypanosoma brucei gambiense represented 0.81% (13/1596) of them, confirming the circulation of human infective parasite in the Fontem focus. Genetic characterization of the 75 T. brucei samples using five microsatellite markers revealed not only multiple T. brucei genotypes (47%), but also single genotypes (53%) in the mid-guts of the wild tsetse flies. These results show that there is a wide range of trypanosome genotypes circulating in the mid-guts of wild tsetse flies from the Fontem sleeping sickness focus. They open new avenues to undertake investigations on the maturation of multiple infections observed in the tsetse fly mid-guts. Such investigations may allow to understand how the multiple infections evolve from the tsetse flies mid-guts to the salivary glands and also to understand the consequence of these evolutions on the dynamic (which genotype is transmitted to mammals) of trypanosomes transmission.