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Members of the Culex pipiens complex are considered as biting nuisance and vectors of important arbo-viruses including West Nile virus (WNV). To analyze the genetic structure of urban and rural populations of Cx. pipiens form pipiens and gain insights into behavioral implications, mosquitoes were collected from established WNV transmission foci in Connecticut from October 2006 through October 2007, examined by using microsatellite markers, and compared with other populations from neighboring states in the northeastern United States. The mean numbers of alleles per locus for the aboveground Cx. pipiens form pipiens populations ranged from 11.5 ± 2.3 to 13.2 ± 2.4 and were not significantly different. In contrast, Cx. pipiens form molestus had greatly reduced allelic diversities with an average of 4.4 ± 1.2 alleles per locus, which was significantly lower than that of any of the Cx. pipiens form pipiens populations analyzed. We did not detect significant genetic differences between urban and rural populations of Cx. pipiens form pipiens from Connecticut nor did we observe temporal genetic changes. However, in a comparative analysis with populations of neighboring states, New Jersey, New York, and Massachusetts, genetic variations associated with geographic distance were identified. In the analyses of Bayesian clustering and principal component analysis, we identified two clusters separating Cx. pipiens form molestus from Cx. pipiens form pipiens populations, indicating that Cx. pipiens form molestus was genetically distinct from any of the Cx. pipiens form pipiens populations examined during this study.