نبذة مختصرة : Abstract Background African trypanosomiasis, caused by protozoa of the genus Trypanosoma and transmitted by the tsetse fly, is a serious parasitic disease of humans and animals. Reliable data on the vector distribution, feeding preference and the trypanosome species they carry is pertinent to planning sustainable control strategies. Methodology We deployed 109 biconical traps in 10 villages in two districts of northwestern Uganda to obtain information on the apparent density, trypanosome infection status and blood meal sources of tsetse flies. A subset (272) of the collected samples was analyzed for detection of trypanosomes species and sub-species using a nested PCR protocol based on primers amplifying the Internal Transcribed Spacer (ITS) region of ribosomal DNA. 34 blood-engorged adult tsetse midguts were analyzed for blood meal sources by sequencing of the mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) genes. Results We captured a total of 622 Glossina fuscipes fuscipes tsetse flies (269 males and 353 females) in the two districts with apparent density (AD) ranging from 0.6 to 3.7 flies/trap/day (FTD). 10.7% (29/272) of the flies were infected with one or more trypanosome species. Infection rate was not significantly associated with district of origin (Generalized linear model (GLM), χ2 = 0.018, P = 0.895, df = 1, n = 272) and sex of the fly (χ2 = 1.723, P = 0.189, df = 1, n = 272). However, trypanosome infection was highly significantly associated with the fly’s age based on wing fray category (χ2 = 22.374, P
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