The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is a perennial insect pest of cultivated maize that was inadvertently introduced into North America in the early 1900s, but population densities have decreased since the widespread adoption of transgenic hybrids that express Bacillus thuringiensis (Bt) toxins. The native American lotus borer, Ostrinia penitalis (Lepidoptera: Crambidae), is among the most ancestral species described in the genus Ostrinia, and has a geographic range that coincides with that of O. nubilalis across major maize growing regions of North America. Due to the recent decrease in O. nubilalis populations, O. penitalis has become more pronounced in light trap samples intended to monitor O. nubilalis. A molecular tool based on variation in restriction endonuclease digestion pattern of a polymerase chain reaction amplified fragment of the mitochondrial cytochrome c oxidase subunit I (coxI) gene was developed and validated to differentiate these two species. This method was applied to light trap samples over a 2-yr period and achieved accurate quantification of species, and shows that O. penitalis can be prevalent in O. nubilalis first flight sampling. These methods are useful for contemporary O. nubilalis field research in North America.