Mitochondrial DNA (mtDNA) from 99 meadow voles (Microtus pennsylvanicus) collected in 13 localities and from 2 Townsend's voles (M. townsendii) from a single locality was assayed for restriction fragment length polymorphism (RFLP) with 13 restriction endonucleases. There was evidence of extensive mtDNA sequence heterogeneity within and among meadow vole populations. Thirty-eight different mtDNA composite phenotypes were found. Two common mtDNA composite phenotypes were shared among five populations, the other mtDNA composite phenotypes being characteristic of individual populations. Estimates of nucleon diversity (ĥ) were high (0.303–0.893), as were measures of intrapopulational nucleotide divergence (px values ranged from 0.0 to 0.038). Population fluctuations and periodic dispersal are the likely mechanisms maintaining high mtDNA composite phenotype diversity in meadow vole populations. Overall interpopulational nucleotide divergence (pxy) was also high (values ranged from 0.007 to 0.045). Cluster analysis clearly separates M. pennsylvanicus and M. townsendii and suggests the separation of the meadow vole populations into eastern and central groups, but there is little evidence of structure within the regional groups. Based on the zoogeography of the populations sampled, the mtDNA RFLP data support the differentiation of Microtus pennsylvanicus into at least two previously described subspecies.