Nucleotide sequence differences in the acetylcholinesterase gene Ace.2 were used to develop an assay to distinguish among North American mosquitoes of the Culex pipiens complex. Taxon-specific polymerase chain reaction primers based on sequence differences within intron 2 of Ace.2 distinguish among the sibling species Cx. pipiens Linneaus and Cx. quinquefasciatus Say and their F1 hybrids. This assay may be used to confirm the species composition of mosquito pools, identify individual specimens collected in arbovirus surveillance programs and other mosquito studies, and define zones of hybridization.