The genetic variability of seven European corn borer populations, Ostrinia nubilalis, from North America and Europe was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing. The nuclear ribosomal internal transcribed spacer 1 (ITS-1) region (approximately 500 base pair [bp]) and four mitochondrial (mtDNA) regions (1550 bp total) were examined. The smartweed borer, Ostrinia obumbratalis, and south-Western corn borer, Diatraea grandiosella, were used for comparisons. Of 106 restriction sites identified (80 in mtDNA and 26 in ITS-1), none differentiated geographical populations, pheromone races, or voltine ecotypes of the European corn borer. The lack of variation in the ITS-1 of European corn borer was confirmed by DNA sequence analysis. The genetic similarity of European corn borer populations, despite their wide geographical range and physiological differences, may be explained by a relatively recent origin for the voltinism and pheromone races, gene flow among races, and/or expansion from genetic bottlenecks.