Western corn rootworm, Diabrotica virgifera virgifera LeConte, and northern corn rootworm, D. barberi Smith and Lawrence, are sympatric species and serious pests of corn cultivation in North America. Comparison of nucleotide sequence of mitochondrial cytochrome oxidase I and II was used to design polymerase chain reaction (PCR) primers that discriminate immature stages of the two species based on differences in amplicon size. Multiplex PCR can be used to give a positive test for each species in a single amplification reaction. This provides a method to identify field caught larvae and facilitates investigations of larval interaction and competition between the species.