We sequenced two regions of the mitochondrial genome, ND4 and d-loop, from 64 Eumeces septentrionalis to assess intra- and interspecific population differentiation. We constructed haplotype genealogies for nine Eumeces septentrionalis septentrionalis populations in previously glaciated regions and used nested clade analysis to examine the role of vicariance and dispersal in shaping the population structure of E. s. septentrionalis in the northern part of its range following Pleistocene paleoclimatological events. In addition, we used DNA sequence data to compare populations of the northern subspecies (E. s. septentrionalis) with the southern subspecies (Eumeces septentrionalis obtusirostris) to determine whether specific level differentiation is evident. For populations of E. septentrionalis in previously glaciated areas, nested clade analyses revealed isolation by distance with restricted gene flow at both haplotype and upper clade levels as the inferred geographical pattern reflecting the lack of overlapping haplotypes in distant populations. These results suggest that colonization of E. septentrionalis into previously glaciated areas was from a single source with restricted gene flow. These results do not support past population fragmentation or colonization from multiple, genetically distinct source populations. Parsimony and maximum likelihood phylogenetic analyses showed reciprocal monophyly between northern (E. s. septentrionalis) and southern (E. s. obtusirostris) subspecies with uncorrected sequence divergence ranging from 6.7–7.0%. These results, combined with the morphological differences found in previous studies, suggest that these allopatric populations are on separate evolutionary trajectories.