Mitochondrial DNA sequence data for the cytochrome-b gene was used to assess genetic divergence among samples of Sorex palustris (Water Shrew) from eastern and western North America. Phylogenetic analysis revealed the presence of three previously observed clades—Cordilleran (S. p. navigator and S. p. brooksi), Coastal (S. bendirii), Boreal (S. p. palustris)—along with a novel Eastern clade (S. p. gloveralleni and/or S. p. albibarbis). Intraspecific divergence between the Boreal and Eastern clades was 3.42%, exceeding interspecific divergence between the Cordilleran and Coastal clades (3.35%). Application of the genetic species concept to the results of this study suggests that the Boreal and Eastern clades of Water Shrews warrant further investigation for recognition as two distinct sister species (i.e., S. palustris and S. albibarbis, respectively). Additional samples and analysis of nuclear markers will be required to substantiate this proposed taxonomic revision.