The mitochondrial DNA (mtDNA) from the Townsend's vole, Microtus townsendii, was compared with mouse mtDNA by positioning vole restriction enzyme fragments on the known laboratory mouse mtDNA molecule by homology hybridization. The vole mitochondrial genome is conserved in general sequence organization and size but does contain a nonhomologous region of more than 600 base pairs not found in the mouse sequence. Thirty-five voles were collected from seven different localities throughout the range of the species, including insular populations on Bowen and Vancouver islands. The variation of vole mtDNA sequences within this species was assayed with six hexameric and four tetrameric type II restriction endonucleases. These individuals can be divided into seven distinct maternal lines. The level of nucleotide substitution between populations is shown to be as high as 0.896 ± 0.350%. The voles from Bowen Island showed no detectable variation within their population, or divergence from a mainland population on the adjacent coast. This fact suggests a recent colonization of Bowen Island. The samples from Vancouver Island fall into two major maternal lines, which show 0.453 ± 0.240% divergence. These insular maternal lines are 0.677 ± 0.257% divergent from the most closely related mainland population. These results suggest that the voles on Vancouver Island represent long-established maternal lines, and are not derived by a recent colonization from a mainland source population. Based on conservative estimates for rates of nucleotide substitution, Vancouver Island has been inhabited by the Townsend's vole since at least the Olympic interglacial.