Abstract Objective Trypanosomosis is a disease of domestic animals and humans resulting from infection with parasitaemic protozoa of the genus Trypanosoma transmitted primarily by tsetse flies. A cross-sectional study was conducted from January-March 2018, to estimate the infection rate of trypanosome in Glossina tachinoides, their distribution, magnitude and involved trypanosome species in Limmu Kosa District of Jimma zone. Results Study methodology involved entomological survey using monoconical traps to study the magnitude of Fly density Flay/Trap/Day (FTD) and tsetse fly dissection to estimate infection rate of trypanosome in vector flies. The study result indicated that there was only one species of Tsetse fly Glossina tachinoides detected with FTD = 4.45. From the total of (n = 284) dissected Glossina tachinoides flies only (n = 5) positive for Trypanosome resulting in 1.76% Infection Rate. Peak trypanosome infections were observed in female tsetse 2.04%, n = 4 and 1.14%, n = 1 in males. Furthermore, 1.06% of Glossina tachinoides were infected by Trypanosome vivax and the remaining 0.70% was Trypanosome congolense. Finally, the study concluded with the recommendation of control and suppression of the vector and parasite was mandatory due to Pathogenic Animal Trypanosomosis.