As the single most important pest of field corn, Zea mays L., throughout most of the Corn Belt, the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), has undergone repeated selection for resistance to a variety of insecticides that persist widely among Nebraska populations. In this study, we used 11 microsatellite markers to genotype two populations with high levels of resistance to methyl-parathion and aldrin (Polk and Stromsburg), two populations with low and intermediate levels of resistance (Mead and Clay Center) from Nebraska, and one population from outside the Corn Belt (Safford, AZ). The genetic diversity measured by observed heterozygosity (H0) was reduced 15–32% in the highly resistant populations compared with the more susceptible populations in Nebraska. Significant genetic differentiation was detected between the resistant and susceptible populations (Polk and Stromsburg versus Mead and Clay Center) in Nebraska (F_ST = 0.016) and between all the populations from Nebraska and Arizona (F_ST = 0.059). The average observed heterozygosities in the populations were positively correlated with insecticide susceptibility based on mortality at diagnostic concentrations of aldrin and methyl-parathion, respectively. These results indicate that the insecticide selection from exposure to aldrin and methyl-parathion may be a contributing factor in shaping the genetic structure of western corn rootworm populations in Nebraska. Factors including isolation by distance and a Wolbachia-induced breeding barrier may have contributed to differentiation of rootworm populations from Nebraska and Arizona.