Previous research has identified a bimodal emergence of apple maggot, Rhagoletis pomonella (Walsh), in the southern Appalachia region of North and South Carolina. Peak emergence of the two groups is separated by a 4-6 week period, with the first emergence occurring in late May to early June, and the second from mid July through August. Laboratory studies suggest that early emergers require a shorter post-diapause developmental period than late emergers. To determine if these different apple maggot populations could be differentiated genetically, the COI and COII genes of mitochondrial DNA of individuals from each group were sequenced and subjected to nested clade and neighbor joining analyses. Apple maggot populations from Michigan, Massachusetts and West Virginia were also included in the analysis for outgroup comparisons.