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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing analyses were used to characterize an amplicon of ~625 bp in 4 of the 5 nominate species of Muscidifurax Girault & Sanders, pupal parasitoids of muscoid flies. A single polymorphic nucleotide site was observed among 2 samples of M. raptor Girault & Sanders. No sequence variation was observed among 3 samples of M. raptorellus Kogan & Legner. The sequence of M. uniraptor Kogan & Legner was identical to that of M. raptorellus. Nucleotide divergence among the Muscidifurax spp. ranged from 0.14 to 0.18 substitutions per nucleotide. Muscidifurax zaraptor Kogan & Legner exhibited multiple haplotypes, 2 of which were characterized by sequencing and 4 others by PCR-RFLP. The sequenced haplotypes differed by 0.08 nucleotide substitutions per site. Restriction site analysis indicated that nucleotide divergence ranged from 0.03 to 0.10 among all 6 haplotypes. Analysis of progeny from individual females indicated that the observed variation in M. zaraptor was caused by multiple haplotypes within individuals rather than differentiation among individuals. These results bring to question the specific status of M. uniraptor and indicate that the genus is native to the Western Hemisphere, and not introduced with their primary host, Musca domestica L, as previously proposed. Heteroplasmy and translocation of aportion of the mitochondrial genome to the nuclear genome are discussed as possible causes for the variation observed in M. zaraptor.