The progressive loss and fragmentation of longleaf pine forests throughout the southeastern United States has led to severe declines in habitat specialists such as the federally listed gopher tortoise Gopherus polyphemus. However, little is known of the range-wide historical genetic structure of this species or how habitat loss may have impacted the genetic diversity of this species over time. As genetic data are lacking in many parts of the gopher tortoise range, management of federally listed populations in Louisiana and Mississippi have been based on conservation units delineated by rivers. However, this current management strategy has been formulated without any knowledge of how riverine barriers might relate to genetic population structure. Here, we examine variability of mitochondrial and nuclear microsatellite markers in gopher tortoise populations across their natural range in the southeastern United States. Results from the mitochondrial data showed a historical phylogenetic break that is coincident with the Apalachicola River and a novel population assemblage located within western Georgia. Genetic structure based on microsatellite loci revealed high levels of genetic differentiation in some regions and a pattern of isolation by distance at two spatial scales. We also find that rivers across the federally listed portion of the range, do not significantly influence gene flow. Coalescent analyses of population demographic change in gopher tortoises indicate evidence of an ancient and severe population decline at sites across the range. Findings from this study provide important information that will aid in the identification of distinct population units for management and in the formulation of recommendations for the future maintenance of genetic diversity in this species.