The Rocky Mountain wood tick, Dermacentor andersoni Stiles, 1908, is of medical and veterinary importance because it can transmit pathogenic agents to humans, domestic livestock, and wildlife. The preferred attachment sites of D. andersoni adults and their ability to induce paralysis in hosts vary among populations, which may have a genetic basis. In this study, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analyses and DNA sequencing were used to determine the genetic variation in the 16S mitochondrial DNA gene of two D. andersoni populations from the Canadian prairies: Saskatchewan Landing Provincial Park, Saskatchewan, and Lethbridge, Alberta. Five haplotypes were detected in each population, but this was considerably lower than the 14 haplotypes reported in a previous study of a laboratory colony of D. andersoni originating from the Rocky Mountains in Montana. In addition, the Canadian populations did not share any haplotypes with the population from Montana. Differences in the genetic composition of the two Canadian prairie populations of D. andersoni compared with the montane population in the United States may have arisen through geographical isolation. These genetic differences between tick populations may also have important implications with respect to their ability to transmit pathogens to hosts. Further studies are needed to determine the extent of genetic variation and the vector potential of ticks from different populations throughout the range of D. andersoni in the United States and Canada.