Nasonia (Hymenoptera: Pteromalidae) is a genus of parasitoid wasps, which is fast emerging as a model system for evolutionary, genetic, developmental and host-endosymbiont interaction studies. In this study, we report a new species, Nasonia oneida, distinguish its behavioral, genetic and morphological features, and characterize its pre-mating and post-mating isolation with the other Nasonia species. Phylogenetic analyses indicate that N. oneida is the sister species to Nasonia giraulti with its own uniquely distinct cuticular hydrocarbon profiles, behavioral characteristics and subtle morphological differences. An important characteristic of N. oneida is the strong mate discrimination shown by the females against all the other Nasonia species. A genetic analysis of this phenotype by interspecies hybridization indicates that this strong discriminating phenotype is recessive. A formal species description of N. oneida Raychoudhury & Desjardins is also provided.