DNA sequence of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), mitochondrial cytochrome c oxidase I (cox1) and II (cox2) genes were characterized and used for population genetic analysis. Twenty-six point mutations were identified from a 2,156 bp DNA sequence alignment. The frequency of polymorphic cox1 DdeI and HaeIII, and cox2 Sau3AI and MspI restriction sites were determined from 1,414 individuals by polymerase chain reaction restriction fragment length polymorphism. Ten haplotypes were observed. A single haplotype was present among 90% of individuals examined, and a HaeIII haplotype was not present in samples from the Atlantic coast. Significant genetic differentiation existed between Atlantic coast and midwestern United States samples, and between sympatric uni- and bivoltine ecotypes. These genetic markers identify regional and ecotype differences in the North American O. nubilalis population.