The genus Dermacentor is represented by 12 species in the New World. We sequenced a 300-bp portion of the mitochondrial 16S ribosomal DNA gene for 28 individual ticks representing 9 of these species and analyzed their phylogenetic relationships. Maximum parsimony, distance (neighbor-joining), and maximum likelihood were all used to resolve tree topologies. Eleven specimens of Dermacentor hunteri Bishopp representing populations across the tick's entire geographic range showed negligible genetic variation, with only single base-pair differences between each of 5 haplotypes. We found high degrees of bootstrap support (66-86%) for monophyly of the genus, but variable support for monophyly of species within the genus. D. hunteri, D. occidentalis Marx, and D. variabilis (Say) each resolved as a monophyletic taxon (79-99% support). D. andersoni Stiles and D. parumapertus Neumann formed a paraphyletic clade (99% support). D. albipictus Packard showed substantial intraspecific variation and warrants further investigation. D. imitans Warburton was distinct from all other Dermacentor spp. on all trees.