Tsetse fly infestation in national parks is a major health risk to both the wildlife and tourists coming to sub-Saharan Africa. However, information on distribution and diversity of tsetse flies and trypanosome infection rate in Protected Areas like Old Oyo National Park in south-west Nigeria is largely unknown. Thus, the study evaluates distribution and
diversity of tsetse flies in Magurba Range of Old Oyo National Park. Twelve Nzi traps were set at 50 m equidistance to capture Glossina species for a period of six months between February and August, 2019, considering both the altitudinal and ecological significance. A total of 136 tsetse flies belonging to four species; G. palpalis, G. tachinoides, G.
morsitans and G. fusca; were captured. More Glossina species were captured during dry season 77.9% (70.0-84.6) than the wet season 22.1% (15.4-30.0). There was significant difference (p = 0.0001; x² = 84.9; OR = 12.5) between the proportion of Glossina species captured at the riverine areas (106; 77.9%) and the woodland/forest areas (30; 22.1%). Glossina captured at ground level and 30 cm above ground were 71 (52.2 %) and 65 (47.8%) respectively. The overall prevalence of trypanosome infection (2.94%) was observed for Glossina spp. The presence of infected Glossina spp. indicated an urgent need to establish a concise strategic vector control in National Parks, in order to reduce the risk of transmission to both wildlife and humans in the area. The park is frequently visited by tourists, rangers, researchers and students for educational purposes.