Genetic diversity was studied at allozyme loci in two Palearctic and one Nearctic population of Aedimorphus (=Aedes) vexans, a species of public health and veterinary importance. The population from Serbia was the most polymorphic (P = 35%) with the highest observed heterozygosity (H_o = 0.027). The lowest observed heterozygosity (H_o = 0.010) was obtained for the Nearctic population. All analyses based on individual (STRUCTURE analysis) and population level (pairwise F_ST, Nm values, AMOVA, Nei's D value) revealed significant structuring between Nearctic and Palearctic populations, indicating a lack of gene flow and thus, the presence of independent gene pools. Taxon-specific alleles at the diagnostic Ao, Hk-2, Hk-3, Hk-4, Idh-1, and Idh-2 loci were used for identification and separation of Nearctic and Palearctic populations. Population genetics study provided valuable information on the correct distinction of Am. vexans populations and their adaptive potential that could find a future use in the studies of vector competence and development of vector-control strategies.