Mosquitoes in the Culex pipiens complex, Cx. p. pipiens L. and Cx. p. quinquefasciatus Say are morphologically similar and important vectors of West Nile and St. Louis Encephalitis viruses in the United States. The subspecies differ with respect to overwintering strategies, with Cx. p. pipiens entering diapause in response to winter conditions and Cx. p. quinquefasciatus lacking this ability, yet they hybridize when found in sympatry. Specimens (n = 646) were collected using gravid traps set along a transect from New Orleans, LA, to Chicago, IL. Microsatellite markers were used to: genetically characterize subspecies and hybrids, determine the degree and extent of hybridization to better define the hybrid zone, and examine the efficacy of hybrid detection between microsatellites and a single-gene assay based on the acetylcholinesterase.2 gene (HotAce.2). The results support the presence of two distinct genetic entities, with abroad, stable hybrid zone in between. Admixture analyses classified >40% of individuals as hybrids. Allelic richness was markedly different at the northern and southern ends of the transect, and there was a significant isolation by distance effect. The hybrid zone seems to be wider and extends further to the south than previous work indicated, and as a result, we propose new boundaries compared with those indicated by a previous study. Microsatellites detected more hybrids than the HotAce.2 assay, although the latter assay remains useful as a screening tool. We suggest that the parental subspecies and the hybrid zone are maintained by heterosis combined with selection for diapause at northern latitudes.