A cross-sectional study was undertaken to assess the prevalence of bovine trypanosomosis in some tsetse-infested and tsetse-free areas of Ethiopia. From August 2010 till April 2011, a total of 1524 animals were parasitologically examined and compared by the haematocrit centrifugation technique (Woo test) and polymerase chain reaction (ITS-1 PCR). The ITS-1 PCR was more sensitive and more accurate in species identification than the Woo test. In ITS-1 PCR, an overall trypanosome prevalence of 31.0% was observed that is significantly (P<0.001) higher than in the Woo test (5.3%). Trypanosoma vivax was the predominant taxon (24.9%), followed by T. theileri (6.0%), T. congolense (2.9%) and Trypanozoon (1.6%). Mixed infections were quite common (14% of all infections). The overall prevalence of trypanosome infections in tsetse area (32.4%) was not different from non-tsetse area (30.5%) neither were the prevalences of T. vivax in both areas (respectively 22.6% and 25.7%). With these high prevalences, bovine trypanosomosis continues to hinder animal production and productivity in Ethiopia, both in tsetse-infested and non-infested parts of the country. Attempts to control African trypanosomosis should also pay attention to mechanically transmitted pathogenic trypanosomes and should adopt the most advanced molecular tests for species identification.