Classical population genetic analyses were used to investigate populations of the western corn rootworm, Diabrotica virgifera virgifera LeConte, in Croatia in 1996 and 2009. The number of alleles was low in both 1996 and 2009; however, more alleles were found in the putative populations surveyed in 2009. Croatia had only 51% of the alleles recorded from the United States and 69% from Europe. However, 10 private (unique) alleles were found in Croatia, which were not found previously in Europe. Most populations were out of Hardy—Weinberg equilibrium, although no linkage disequilibrium was found. Low to no genetic differentiation was found between population pairwise comparisons in 1996, with a greater level of differentiation found between populations sampled in 2009. Using the program STRUCTURE, a single genetic cluster was found for populations sampled in 1996 and 2009. However, two genetic clusters were detected when the 1996 and 2009 data were combined, indicating significant temporal differentiation. Isolation by distance pattern of gene flow characterized populations sampled in 2009 only when the most distant population of Ogulin (the head of the expansion front) was included in the analysis. When Ogluin was excluded from the 2009 analysis no isolation by distance pattern was found. The possible impact that control practices have had on the population genetics of D. v. virgifera in Croatia from 1996 to 2009 are discussed in light of the temporal genetics differences found.