Many vertebrate taxa show genetic differentiation between populations in northern and southern California. This genetic pattern may reflect a common environmental history for these species. For example, a previous study of the California vole (Microtus californicus) showed morphological divergence between populations in northern and southern California and decreased fertility in crosses between the populations. To investigate phylogeographic differences in this species, we assessed variation in mitochondrial and nuclear DNA throughout much of its distribution from Oregon to Baja California. We generated molecular data (mitochondrial cytochrome b and nuclear acid phosphatase V intron [AP5]) for 178 individuals. Examination of these data suggests 2 phylogeographic groups that are largely discordant with the boundaries of 17 currently recognized subspecies. Estimates of pairwise genetic divergence between these groups for cytochrome b are as high as 4.46% uncorrected p. Sequence data for AP5 also indicate a division between populations of M. californicus in northern and southern California. Examination of data from the mitochondrial and nuclear markers together suggests limited gene flow between clades. These data are concordant with other studies that suggest that mountain ranges in California were important in within- and possibly between-species divergence and subsequent contact. The general distribution of each clade, combined with a geographic information system analysis of known capture sites, suggests that clade divergence may be correlated with ecological differences. Our study creates a new framework for reevaluating morphological and ecological diversity in this species, and with more diverse markers, possibly the recognition of 2 species of California voles.