Aedes vexans (Meigen), the vexans mosquito, is a species that prefers mammalian hosts and is a vector of West Nile virus (family Flaviviridae, genus Flavivirus). It is one of the most widespread pest mosquitoes in the world and North America, and it is commonly found in southern Canada and continental United States. Population structure of this species in Kansas was examined using DNA sequences of a 423-bp region of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene, relative to three other states. From the 54 Kansas samples, a total of 39 nucleotide positions were polymorphic, with 34 haplotypes. Of the 34 haplotypes, 22 (79%) were not shared among populations. The average haplotype diversity (0.953) from 11 Kansas populations indicated a high level of genetic diversity in Ae. vexans. Among the Kansas, South Dakota, Texas, and Louisiana samples, a total of 40 haplotypes were observed. Analysis of molecular variance was conducted on the resulting haplotypes for all populations and no geographical structure was observed among populations by using isolation-by-distance tests. This first genetic study of Ae. vexans provides evidence that there is a large amount of haplotype variation within and among populations, and gene flow occurs across broad geographical areas in this species.