AbstractOver 10000Glossina pallidipestsetse flies were collected during two field studies in the Zambezi Valley, Zimbabwe and one in the Luangwa Valley, Zambia. These were screened for mature trypanosome infections and 234 dot‐blot preparations were made of infected midguts, which were screened using DNA probes or PCR with primers specific to different species or types of the trypanosome subgenusNannomonas. Over 70% of midgut infections were successfully identified as eitherTrypanosoma godfreyi, T. simiaeor three types ofT. congolense, savannah, riverine‐forest and Kilifi. The relative abundance of species and types did not vary significantly between study locations, habitat, season or tsetse age or sex, although there were differences between DNA probe and PCR results. Mixed species and/or mixed type infections were common and were more often detected using PCR. The distribution of infections among flies was highly aggregated, but there was no tendency for multiple infections to accumulate in older flies, implying that sequential superinfection may be uncommon. Possible explanations for these patterns are discus