Baseline surveys were conducted in the Upper West Region of Ghana to assess the distribution and densities of tsetse species, as well as the prevalence of bovine trypanosomosis. The entomological survey was designed to cover the suitable tsetse habitats along the three main rivers in the study area (i.e. Black Volta, Kulpawn and Sissili). Results indicated the presence of Glossina tachinoides in all three river basins, whilst Glossina palpalis gambiensis was only found close to the southern limit of the study area. A random sampling of 1800 cattle of the West African Short Horn, Sanga and Zebu breeds from 36 randomly selected grid cells covering the study area showed substantial differences between parasitological and serological prevalences. The average parasitological prevalence was estimated at 2.5% (95% CI: 1.06–5.77) with the majority of the infections due to Trypanosoma vivax. Most of the infected cattle were found close to the major river systems. The serological prevalence, measured using Enzyme Linked Immunosorbent Assay (ELISA), test was 19% (95% CI: 14.03–25.35). Cattle with anti-trypanosomal antibodies were also found throughout the study area.