Background Tsetse 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 in Glossina species from Cameroon, Chad and Nigeria.Results Tsetse flies were collected from five different locations and dissected. DNA was extracted and polymerase chain reaction PCR was used to detect the presence of Sodalis glossinidius, Spiroplasma sp and Wolbachia using 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%) and Glossina 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 (Wolbachia and Spiroplasma). The overall symbiont prevalence was 0.88%, 3.66% and 11.00% respectively, for Sodalis, Spiroplasma and Wolbachia. Prevalence varied between countries and tsetse species. No Spiroplasma was detected in samples from Cameroon and no Sodalis was found in samples from Nigeria.Conclusion The present study revealed for the first time, the presence of infection by Spiroplasma in 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.