BackgroundTsetse flies are cyclical vectors of African trypanosomiasis. They have established symbiotic associations with different bacteria, which influence certain aspects of their physiology. The vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by various factors, amongst which are bacterial endosymbionts. Symbiotic interactions may provide an avenue for the disease control. The current study provided the prevalence of 3 tsetse symbionts inGlossinaspecies from Cameroon, Chad and Nigeria.ResultsTsetse flies were collected from five different locations and dissected. DNA was extracted and polymerase chain reaction PCR was used to detect the presence ofSodalis glossinidius, Spiroplasmasp andWolbachiausing specific primers. A total of 848 tsetse samples were analysed:Glossina morsitans submorsitans(47.52%),Glossina palpalis palpalis(37.26%),Glossina fuscipes fuscipes(9.08%) andGlossina tachinoides(6.13%). Only 95 (11.20%) were infected with at least one of the 3 symbionts. Among the infected, 6 (6.31%) were carrying mixed infection (WolbachiaandSpiroplasma). The overall symbiont prevalence was 0.88%, 3.66% and 11.00% respectively, forSodalis, SpiroplasmaandWolbachia. Prevalence varied between countries and tsetse species. NoSpiroplasmawas detected in samples from Cameroon and noSodaliswas found in samples from Nigeria.ConclusionThe present study revealed for the first time, the presence of infection bySpiroplasmain tsetse in Chad and Nigeria. These findings provide useful information to the repertoire of bacterial flora of tsetse flies and incite to more investigations to understand their implication in the vector competence of tsetse flies.