The increase of human population, combined with climatic changes, contributed to the modification of spatial distribution of tsetse flies, main vector of trypanosomiasis. In order to establish and compare tsetse presence and their relationship with vegetation, entomological survey was performed using biconical traps deployed in transects, simultaneously with phyto-sociological study, on the Comoe river at its source in the village of Moussodougou, and in the semi-protected area of Folonzo, both localities in Southern Burkina Faso. In Folonzo, the survey revealed a diversity of tsetse with 4 species occurring with apparent densities as follows: Glossina tachinoides (8.9 tsetse/trap/day); G. morsitans submorsitans (1.8 tsetse/trap/day); G. palpalis gambiensis (0.6/trap/day) and G. medicorum (0.15 tsetse/trap/day). In Moussodougou, a highly anthropized area, mainly G. p. gambiensis was caught (2.06 tsetse/trap/day), and rarely G. tachinoides. The phyto-sociological study allowed discrimination of 6 types of vegetation in both localities, with 3 concordances that are riparian forest, shrubby and woody savannah. In Moussodougou, all tsetse were caught in the riparian forest. That was also the case in Folonzo where a great proportion (95 to 99 % following the season) of G. p. gambiensis and G. tachinoides were caught in the gallery, while G. m. submorsitans was occurring as well in the gallery as in the savannah, and G. medicorum in the forest gallery. This study showed that although G. tachinoides and G.p. gambiensis are both riparian, they do not have the same preference in terms of biotope.