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Genetic variation in the nuclear rDNA ITS1 region of western corn rootworm, Diabrotica virgifera virgifera (WCR), and Mexican corn rootworm, D. v. zeae (MCR) was studied. Two sites were detected which differentiated WCR and MCR in the 642-base sequence. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the first internal transcribed spacer region (ITS1) sequence revealed no variation within or among the twelve WCR and two MCR populations. PCR-RFLP of 75% of the mitochondrial DNA genome detected one significant polymorphic site out of the approximately 190 restriction sizes observed in WCR. The polymorphism did not differentiate geographical populations of WCR and is not diagnostic for the subspecies. The low levels of variation observed in WCR suggests either high levels of gene flow or a recent geographical expansion from a relatively small base. Gene flow would facilitate the rapid spread of traits that could compromise control programs, such as insecticide resistance or behavioral modifications. The minimal genetic differentiation between WCR and MCR raises questions about the evolutionary history of these subspecies and how the distinct phenotypes are maintained.