The geology of the Pleistocene, and particularly the Last Glacial Maximum approximately 26.5 ka, is a critical driver of species’ present-day distributions and levels of genetic diversity in northern regions. Using mitochondrial DNA sequence data, we tested several predictions relating to the postglacial recolonization of the northern United States and southern Canada by Mudpuppies (Necturus maculosus). Our analyses revealed a significant split between western and eastern lineages, with the divide corresponding to the location of the Mississippi River. Our data support the presence of one or more Mississippian glacial refugia, with subsequent expansion and diversification of a western cluster into the upper Midwest, and an eastern cluster into the eastern Great Lakes and New England. As predicted in cases of postglacial colonization, each of these clusters contains a single widespread and common haplotype, along with numerous low-frequency, closely related haplotypes. Given recent conservation concerns about amphibians in general, and Mudpuppies specifically, we discuss our results in light of species conservation. Knowledge of a species’ genetic diversity allows for informed management and facilitates decisions that preserve local adaptation and evolutionary potential.