To determine and compare the prevalence of trypanosome infections in different livestock species (cattle, pigs and goats) in areas where game animals are scarce and livestock constitute the main food source of tsetse, a survey was conducted on the plateau of the Eastern Province of Zambia in Katete and Petauke districts where Glossina morsitans morsitans is the only tsetse species present. Blood was collected from a total of 734 cattle, 333 goats and 324 pigs originating from 59 villages in both districts and was examined using the buffy coat method and the PCR-RFLP as diagnostic tools. The prevalence of trypanosome infections differed substantially between livestock species. Using microscopic diagnostic methods, trypanosome infections were detected in 13.5% of the cattle and 0.9% of the pigs. All goats were parasitologically negative. The PCR-RFLP analyses increased the trypanosomiasis prevalence to 33.5, 6.5 and 3.3% in cattle, pigs and goats respectively. The majority of the infections (91.2%) were due to Trypanosoma congolense. The presence of a trypanosome infection in cattle and pigs resulted in a significant decline in the packed cell volume. The outcome of the study clearly shows that despite the availability of goats and pigs, cattle seem to be the major livestock species affected by the disease in trypanosomiasis endemic areas. The high proportion of infections in cattle could be partly attributed to their higher availability and attractiveness to tsetse.