Genetic variation of mitochondrial DNA (mtDNA) and the nuclear ribosomal spacer, ITS1, in local and dispersed geographical populations of northern corn rootworm, Diabrotica barberi Smith & Lawrence was examined. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for mtDNA and DNA sequencing plus PCR-RFLP analysis was used for ITS1. Insects were collected in 10 states ranging from Pennsylvania to the Great Plains. Sequencing of the ITS1 amplicon revealed three potential polymorphic sites, one of which altered a restriction site for the restriction enzyme BclI. PCR-RFLP analysis with BclI detected three genotypes. Many beetles had heterogeneity at the nucleotide site recognized by BclI, which was supported by DNA sequence data. There appears to be a phylogeographic pattern of ITS1 genetic diversity. Eastern populations were homogeneous for one genotype, populations from central and northern locations had two genotypes, and western populations were composed of all three genotypes. The mtDNA had 58 haplotypes that displayed a strong east-west geographical partition. The region of overlap occurred in a few counties of east-central Illinois. Populations to the east had less variability than those to the west. A network of restriction site changes and trees based on genetic distance measurements of the mtDNA produced two distinct clades. One clade contained all the eastern haplotypes along with a group of haplotypes from the northern Great Plains. The other clade included the remaining western haplotypes. Possible reasons for this population structure including expansion from different glacial relicts, historic host plant differences, and endosymbiont driven reproductive incompatibilities are discussed.