The evolution of tritrophic interactions requires adaptations of natural enemies to both herbivorous hosts and host foodplants that can ultimately lead to the formation of locally adapted populations, host races, or ecological species. To situate our model organism, Cotesia congregata (Say), within this spectrum, we estimated genetic differentiation of wasps originating from two common host—foodplant complexes, Manduca sexta L. on tobacco (“MsT”) and Ceratomia catalpae (Haworth) on catalpa (“CcC”). To test the hypothesis that C. congregata associated with these host—foodplant, complexes were genetically differentiated even where they occur in sympatry, we experimentally introduced tobacco at an established “catalpa site” for two consecutive years and compared wasps collected at this site with wasps collected from allopatric tobacco and catalpa sites in the mid-Atlantic region of North America. Using seven microsatellite loci and the mitochondrial COI locus, we found differences in microsatellite allele frequencies based on host—foodplant complex (Φ_PT values 0.47–0.50 among populations from different host—foodplant complexes), but not based on geographic location (mean Φ_PT=0.03). COI haplotypes of MsTand CcC wasps exhibited ∼2% sequence divergence, even where the host—food complexes were colocated within meters at the experimental site. Results indicate that MsT and CcC wasps represent distinct genetic lineages that are most likely incipient or nascent species with limited gene flow.