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A taxonomic key, based on restriction enzyme analysis of the second internal-transcribed spacer (ITS-2) in the nuclear ribosomal DNA gene, was developed for identification of 17 Ixodes tick species in the United States. This key includes: Ixodes affinis Neumann, Ixodes angustus Neumann, Ixodes baergi Cooley and Kohls, Ixodes brunneus Koch, Ixodes cookei Packard, Ixodes dentatus Marx, Ixodes jellisoni Cooley and Kohls, Ixodes kingi Bishopp, Ixodes minor Neumann, Ixodes muris Bishopp and Smith, Ixodes pacificus Cooley and Kohls, Ixodes scapularis Say, Ixodes sculpularis Neumann, I. spinipalpis Hadwen and Nuttall, Ixodes texanus Banks, Ixodes uriae White, and Ixodes woodi Bishopp. A 900-bp fragment of the ITS-2 was amplified using the polymerase chain reaction. This fragment was then digested with the restriction enzymes MspI and CfoI, and the digested fragments were size fractionated on a 2.5% high-resolution agarose gel. A dichotomous key was developed based on digested fragment sizes relative to a standard set of size markers. Little intraspecific variation in restriction fragment banding patterns was detected.