African Animal Trypanosomiasis (AAT) is among several constraints hindering development of the livestock sector in Tanzania. A survey was conducted in Liwale district located in southern Tanzania in 2013 to determine the population density of Glossina species, distribution pattern and Trypanosome species infection rate in tsetse flies. A total of 200 flies were collected from the study area and three Glossina species were identified. The proportional abundance of all trapped flies was 90% (180) for Glossina pallidipes, 6% (12) for G. brevipalpis and 4% (8) for G. m. morsitans with apparent densities (fly/trap/day - FTD) of 0.44. Higher density of Glossina pallidipes was observed in villages closer to than those far from the Selous game reserve. Trypanosomes were detected and identified by microscopy and ITS1 polymerase chain reaction (PCR) assay on DNA purified from 200 flies. Glossina pallidipes was the only fly found infected by three Trypanosoma species, namely T. vivax (60%), T. simiae (10%) and T. brucei (30%) with an overall infection rate of 10% (20/200). A higher proportion of trypanosome infections were observed in female tsetse flies than in males. Results of this study show that G pallidipes is the major Glossina species harboring pathogenic trypanosomes in Liwale district and that the Selous game reserve is a potential reservoir of trypanosomes in terms of parasite abundance and species diversity.