Pygmy Shrews in North America have variously been considered to be one species (Sorex hoyi) or two species (S. hoyi and S. thompsoni). Currently, only S. hoyi is recognized. In this study, we examine mitochondrial DNA sequence data for the cytochrome b gene to evaluate the level of differentiation and phylogeographic relationships among eleven samples of Pygmy Shrews from across Canada. Pygmy Shrews from eastern Canada (i.e., Ontario, Quebec, New Brunswick, Nova Scotia, and Prince Edward Island) are distinct from Pygmy Shrews from western Canada (Alberta, Yukon) and Alaska. The average level of sequence divergence between these clades (3.3%) falls within the range of values for other recognized pairs of sister species of shrews. A molecular clock based on third position transversion substitutions suggests that these two lineages diverged between 0.44 and 1.67 million years ago. These molecular phylogenetic data. combined with a reinterpretation of previously published morphological data, are suggestive of separate species status for S. hoyi and S. thompsoni as has been previously argued by others. Further analysis of specimens from geographically intermediate areas (e.g., Manitoba. northern Ontario) is required to determine if there is secondary contact and/or introgression between these two putative species.