Nucleotide sequence information on intemal transcribed spacer (ITS) 1 and ITS 2 regions ofthe nuclear ribosomal DNA multigene family was used to develop a polymerase chain reaction assay thatidentifies Culer nigripeilpas Theobald. The assay uses species-specific forward and reverse primers for C.x.nigripalpus and can be used along with previously described primers to distinguish among 4 common taxa ofCulex (Culex) of the eastern USA with a single thermal cycler program. The assay distinguishes among the 4taxa Cx. nigripalpus, Cx. restuans Theobald, Cx. salinarius Coquillett, and members of the Cx. pipiens Linnaeuscomplex. This assay may be used to verify the morphological identification of individual specimens of Culexor to confirm the species composition of mosquito pools.