Trypanosomes in the dissection-positive proboscis of Glossina pallidipes were identified by PCR using species-specific primers. Of the 3741 flies dissected 643 were proboscis positive. PCR was performed on 406 dissection-positive probosces giving positive identifications in 352 (86·7%) and infection rates of 14·8% for congolense-type infections, 2·8% for vivax- type infections and 1·4% for the unidentified group. Of the 352 PCR identified infections 225 were single, 111 were double, 13 were triple infections and there were 3 quadruple infections. Statistical analysis suggests that mixed infections group into 3 largely separate divisions among the tsetse population (i) Trypanosoma congolense savannah and T. congolense Kenya coast, (ii) T. simiae, T. congolense Tsavo and T. godfreyi and (iii) T. vivax. We conclude that either differing feeding patterns among members of the fly population or the ability of the trypanosomes in each of the infection categories to significantly influence the maturation of trypanosomes in the other categories are the most likely causes of the groupings noted. Chi-squared analysis of dissection and PCR methods of trypanosome identification revealed profound differences (χ = 19·1; D.F. = 1; P > 0·05). If confirmed in other studies these findings have serious implications for our understanding of trypanosome epidemiology in tsetse flies, much of which is founded on data from dissection-based trypanosome identifications.