Although trypanosomatids (Trypanosomatidae: Kinetoplastida) are common inhabitants of dipteran guts, prevalence in natural fly populations has not been studied. We investigated factors associated with trypanosomatid prevalence in eight species of woodland Drosophila (Drosophilidae: Diptera) collected from five sites in southwest Ohio. We collected infected flies from every site, over both years of our study, and from every Drosophila species. Prevalence differed with host species, but not between sites or with host sex. Prevalence was highest in the most abundant species, members of the subgenus Sophophora, species using decaying fruit as breeding sites, and those able to use more than one type of substrate for oviposition.