The Palaearctic parasitoid Microctonus aethiopoides Loan (Hymenoptera: Braconidae) has been introduced to North America for biological control of weevils in the genera Sitona and Hypera (Coleoptera: Curculionidae) and to Australia and New Zealand for control of Sitona discoideus Gyllenhal. Various geographic and host-associated populations of M. aethiopoides have exhibited differences in host preference, host range, and adult morphology. These differences have generally been interpreted as indicative of genetically differentiated biotypes of M. aethiopoides, but direct genetic evidence of biotypic variation has been lacking. Nucleotide sequence data were generated from the gene regions COI, 16S, 28S, and β-tubulin to assess genetic variation among M. aethiopoides reared from various host species collected in Australia, Iran, New Zealand, the United States, and 10 European countries. Ten adult morphological characters were also measured to validate the identity of the specimens and to assess morphological variation among the geographic and host-associated populations. Parsimony and maximum likelihood analyses of the COI, 16S, and β-tubulin sequences provided strong support for the presence of at least two M. aethiopoides biotypes, one associated with Hypera species and the other with Sitona species. There was also evidence for genetic divergence among parasitoids associated with different Sitona species. Morphological variation was also closely correlated with host species, but the occurrence of morphological variation in the absence of genetic variation suggested morphological characters should be used cautiously with M. aethiopoides biotypes.