During two consecutive surveys (February and August/Sept 2002), a total of 970 cattle from the cattle population of Mafia Island (United Republic of Tanzania) were blood-sampled. All blood samples were microscopically screened for the presence of trypanosomes and a portion of these were checked for antibodies with an Ab-ELISA and for the presence of trypanosomal DNA with PCR. Microscopic evidence of trypanosomes of the congolense group (sub-genus Nannomonas) was found in 0.8% of the animals (8/970) and in two cases the species identified was confirmed by PCR as Trypanosoma congolense savannah type. Non-pathogenic Trypanosoma theileri were detected in 3.2% (31/970) of the samples using the Dark Ground-Buffy Coat (DG-BC) technique. For survey 1 (S1), detection of antibodies (Ab-ELISA) against pathogenic trypanosomes indicated a seroprevalence of 14.2% (68/480). Of the samples, either DG positive or with a PCV lower then 25, examined by PCR, a total of 8.4% (5/59) (selected from 970 samples), were found positive for T. congolense. The low prevalence of pathogenic trypanosomes on Mafia Island is intriguing, especially in view of the omnipresence of the tsetse fly Glossina brevipalpis. Although the presence of detected trypanosomal antibodies does not necessarily indicate a current infection, the combination of serological/parasitological examinations and the results of the PCR do support this low prevalence of trypanosomosis in cattle. Despite the low prevalence, pathogenic trypanosomes are present on Mafia Island and possible reasons for this low infection rate, taking account of the relation between Glossina species present, transmission risk and trypanosomes found in cattle, are discussed also in view of a future appropriate intervention strategy.