Documentation of intraspecific genetic lineages and their evolutionary history can provide insight for current and future conservation and management actions. The Blanding’s Turtle, Emys (=Emydoidea) blandingii, is a long-lived species with a relatively narrow latitudinal distribution centered around the Great Lakes, but extending from Nebraska to Nova Scotia. It is listed as endangered or threatened throughout most of its range mainly due to habitat loss. Microsatellite loci have been predominantly used to test and generate hypotheses concerning the number of evolutionarily significant units and the history of lineage diversification in this species. Here we describe haplotypes from two mitochondrial and three nuclear loci generated from 32 localities across the species’ range to provide an additional perspective on these hypotheses. Haplotype and nucleotide diversity were low in both sets of loci, with mitochondrial polymorphism comparable to the lowest found in any North American freshwater turtle. Spatial analyses of population differentiation supported the presence of two lineages with a boundary in eastern Ontario that is roughly associated with the Appalachian Mountains as proposed by Mockford et al (2007). We suggest that the low diversity in these loci is likely related to periodic range contractions and expansions associated with glacial cycles and that the two lineages recovered result from a deeper history of diversification. Our results further support previously identified evolutionary significant units and help to reconcile population structure found at smaller spatial scales, outcomes that will better inform conservation decision making for the species.