Objectives : In order to assess the species composition of tsetse flies among savannah and forested biotopes, an entomological survey was conducted on these insects in the Ndende region (province of Ngounié), ancient focus of Human African Trypanosomiasis (HAT) for identifying the potential sites of the HAT transmission. Methodology and results: A total of 14 Vavoua traps was used with 7 traps installed along the rivers present in the forest gallery and 7 others in the savannah near the villages. The study was done from 23 rd October to 7 th November 2015. In total, 697 tsetse flies were captured (ADT=3.32 tsetse flies/trap/day). The highest abundance of tsetse flies was found in the forest with 678 specimens, while the lowest abundance was observed in the savannah with 19 specimens. The identified tsetse flies are G. fuscipes fuscipes, G. nashi, G. frezili and G. palpalis palpalis. G. fuscipes fuscipes (95%) was the most abundant species. Conversely, G. frezili (1%), G. nashi (2%) and G. palpalis palpalis (2%) were less in number. Conclusion and application: The results of this study showed the presence of four tsetse flies species, biological vectors of HAT, in the Ndendé region. The high abundance of G. fuscipes fuscipes and the presence of G. palpalis palpalis, vectors of HAT, in different savannah and forested biotopes, with strong predominance in forest, suggest a probable risk of transmission of trypanosomes in the Ndende region. Thus, the forested biotope would be the priority areas for implementing a vector control in order to limit the risks of HAT transmission. Therefore, these results require studies aiming at identifying spatial and temporal dynamics of tsetse flies and their role in the eventual transmission of HAT in the Ndendé region, southern of Gabon.