The definition and phylogenetic placement of the autogenous molestus form of Culex pipiens has puzzled entomologists for decades. Renewed research efforts involving this mosquito stem from its unresolved relationship to sister taxa Cx. p. pipiens [L.] and Cx. p. quinquefasciatus Say, and its undefined role in arbovirus transmission. We investigated sequence variation in numerous mitochondrial, nuclear, and nuclear-ribosomal genetic markers from both colony and field-collected Cx. p. pipiens and Cx. p. molestus by polymerase chain reaction, cloning, single strand conformation polymorphism (SSCP), restriction fragment length polymorphism, and sequencing. We identified genetic differences between Cx. p. pipiens and Cx. p. molestus Forskål in the unidentified SH60 Cx. p. pipiens-specific fragment described by Crabtree et al. (1997). SSCP analysis of the SH60 fragment demonstrated a high degree of polymorphism in Cx. p. pipiens collections compared to a single genetic form in corresponding populations of Cx. p. molestus. Cloning and sequencing of this marker revealed that individual Cx. p. pipiens contained as many as four unique SH60 alleles, with some common alleles shared among individuals collected from distant locations. In contrast, Cx. p. molestus individuals each had a single SH60 allele which was also found in corresponding above ground Cx. p. pipiens populations. These preliminary results support the hypothesis that independent genetic bottleneck events may have occurred through which Cx. p. molestus populations were founded from local above ground Cx. p. pipiens populations.