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