Aboveground and belowground populations of the mosquito Culex pipiens L. are traditionally classified as form pipiens and form molestus, respectively, and gene flow between forms is thought to be limited. Relatively few f. molestus populations have been found in the United States, which has hindered their study in North America. In this study, we used microsatellites to characterize a newly discovered population of f. molestus in Chicago, IL, and compared levels of genetic diversity and differentiation in aboveground and belowground populations from Chicago and New York City, NY. Levels of genetic diversity, as measured by expected heterozygosity and allelic richness, were markedly lower in both f. molestus populations. Allele frequencies were distinctly different between the two f. molestus populations, and some alleles were present in one belowground population and not the other. Pairwise F(ST) values between populations indicated that f. molestus populations were highly divergent from each other, as well as from their associated aboveground populations. Cluster analysis suggested the most likely number of groups was three, with the four f. pipiens populations in one cluster, and each of the f. molestus populations in its own cluster. Admixture analysis detected a low number of hybrids, 8%, between forms. We also tested the efficacy of two assays purported to distinguish between the forms, the CQ11 assay and a restriction fragment-length polymorphism assay of the COI gene, and found neither assay reliable in this regard. Our findings support the hypothesis that f. molestus populations in Chicago and New York City arose from local aboveground populations.