Parasitic wasps are among the most speciose of all animals yet the drivers and mechanisms underlying their remarkable diversity are not well understood. Cotesia congregata (Say) and host, Manduca sexta (L.), have long-served as a model system for host-parasite and tritrophic interactions at multiple levels, as well as the evolution of symbiotic coevolved bracoviruses that suppress host defenses to parasitism. We investigated potential barriers to reproduction between two genetically differentiated host-foodplant complex sources of C. congregata originating from caterpillar hosts, M. sexta on tobacco (“MsT wasps”) or Ceratomia catalpae (Boisduval) on catalpa (“CcC wasps”). Both MsT and CcC wasps displayed ∼30% lower responses to cuticular washes of females from the alternate complex. Male courtship songs differed with respect to frequency, absolute amplitude, and duration of song elements. Despite differences in courtship behaviors, MsT and CcC wasps mated and produced offspring in the laboratory. However, mating success of F₁ MsT♂ × CcC♀ wasps was lower and fitness of F₁ CcC♂×MsT♀ hybrids was greatly reduced due to encapsulation and melanization of wasp eggs in M. sexta. Heritable differences in brood size and secondary sex ratios of emergent adults indicate hostspecific adaptations. Overall, results demonstrate the evolution and accumulation of both pre- and postzygotic barriers to reproduction, two of which are asymmetrical, and offer additional support that MsT and CcC wasp are incipient species.