A study was carried out for four years in the forest area of Daloa in Côte d'Ivoire to assess the rate of trypanosome infection in tsetse, and thereby the trypanosomiasis infection risk. In different Glossina biotopes, 18,908 Glossina palpalis palpalis were caught with Vavoua traps and were dissected. The most widespread species of trypanosomes infecting the Glossina was Trypanosoma congolense (7.63%) followed by T. vivax (4.50%). Trypanosoma brucei, the trypanosome responsible for animal and human African trypanosomiasis (HAT), was found only in 34 of the tsetse flies collected, and it had a very low infection rate (0.18%). Although infected tsetse flies were captured in all habitats examined, the infection rate was relatively higher along footpaths (0.44%), in farms (0.20%) and around forested water springs (0.27%) compared to the edge of villages (0.06%) and forest borders (0.05%). Among the 34 tsetse flies infected with T. brucei, only 0.05% had parasites exclusively in their salivary glands. Our results suggest that footpaths, plantations of coffee and cocoa and forested water springs are potential biotopes where the risk of infection by T. brucei is most important. The anthropophily of Glossina associated with the high number of parasites in these sites could be the reason for the disease is endemic in Daloa today. In our study, female Glossina were infected more frequently with trypanosomes (0.14%) than males (0.04%) and generally, females lived longer than males. It is likely that the longevity of females, which carry parasites, is the major cause for the endemicity of HAT in this locality.