Three species of Dermacentor, Dermacentor albipictus, Dermacentor andersoni and Dermacentor variabilis, commonly occur in Canada. D. andersoni and D. variabilis are morphologically similar and are important vectors of human and animal pathogens. A practical polymerase chain reaction (PCR) assay, based on the amplification of part of the second internal transcribed spacer ribosomal DNA (pITS-2 rDNA), was developed to distinguish D. andersoni from D. variabilis. In addition, single-strand conformation polymorphism (SSCP) analysis of the pITS-2 rDNA provided a reliable method of distinguishing specimens of the three species of ixodid tick. PCR and pITS-2 SSCP were also used to determine whether there was hybridization between D. andersoni and D. variabilis at two localities in Saskatchewan where they occur in sympatry. These molecular tools will be useful for the unequivocal identification of D. andersoni and D. variabilis at all life cycle stages, which is essential for studies on their ecology and on the transmission of tick-borne diseases. Also, pITS-2 SSCP may be of potential use for discriminating among the other morphologically similar species within the genus Dermacentor.