Pseudacteon curvatus and P. obtusus are two species of parasitic phorid flies (Diptera: Phoridae), introduced in the southern USA for biological control of Solenopsis fire ants (Hymenoptera: Formicidae). The preference of both parasitoid species (P. curvatus and P. obtusus) for native Solenopsis fire ants (S. geminata complex) versus imported fire ants, S. saevissima complex (S. invicta and S. richteri) was investigated in four-choice olfactometer assays. We hypothesized that differences in the venom alkaloid profiles of fire ant species mediate host preference in phorid flies. In olfactometer bioassays, female P. obtusus showed preference for the venom alkaloid (cis + trans alkaloid mixture) of the imported S. invicta fire ant over the venom alkaloid of the native geminata fire ant. Similarly, P. obtusus showed preference for the venom alkaloid of the S. richteri fire ant over that of the native S. geminata ants. Pseudacteon curvatus also showed preference for the venom alkaloid of the S. invicta and S. richteri fire ants over that of the native geminata ants in separate bioassays. The results suggest that host preference in P. curvatus and P. obtusus is mediated, at least in part, by differences in the venom alkaloid profile of various fire ant species. This finding may explain why Pseudacteon species associated with either the S. geminata or S. saevissima complexes have not switched to the other group despite the overlap of native and imported fire ants in some areas of their distributions.