We assessed the structure and latitudinal selection that might result in sensitivities to critical day-lengths that trigger diapause between Culex pipiens populations distributed along North-South and East-West axes in eastern North America. Strong population structure between Cx. p. pipiens and Cx. p. quinquefasciatus existed. Among Cx. p. pipiens, a 100-km increase in the latitudinal change resulted in an increased square root of FST by 0.002. A 100-km increase in the longitudinal change caused an increased square root of FST by 0.035. A lack of latitudinal influence on the structure between Cx. p. pipiens populations suggests a uniform signal using the 12 microsatellite markers, which might increase the risk of West Nile virus (WNV) transmission toward northern areas because of longer breeding season, extend host-seeking period, and larger population size. Northern Cx. p. pipiens may have undergone additional generations before diapause is triggered, magnifying population size when WNV amplification is peaking.