The gopher tortoise (Gopherus polyphemus) has experienced dramatic population declines throughout its distribution in the southeastern United States and is federally listed as threatened in the area west of the Tombigbee-Mobile Rivers. While there is molecular support for recognizing the listed portion of the range as genetically distinct, other research has suggested that additional population structure exists at both range-wide and regional scales. In this study, we sought to comprehensively define structure at both spatial scales by doubling the data available in terms of the number of sampling sites, individuals and microsatellite loci compared to previously published work. Patterns of genetic diversity, gene flow and demographic history were also compared across the range. We collected 933 individuals from 47 sampling sites across the range and genotyped them for 20 microsatellite loci. Our range-wide analyses supported the recognition of five genetic groups (or regions) delineated by the Tombigbee-Mobile Rivers, Apalachicola-Chattahoochee Rivers, and the transitional areas between several physiographic province sections of the Coastal Plains (i.e., Eastern Gulf, Sea Island, and Floridian). Genetic admixture was found at sampling sites along the boundaries of these genetically defined groups. We detected some degree of additional genetic structure within each of the five regions. Notably, within the federally listed portion of the range, we found some support for two additional genetic groups loosely delineated by the Pascagoula-Chickasawhay Rivers, and we detected four more genetic groups within the Florida region that seemed to reflect the influence of the local physiography. Additionally, our range-wide analysis found the periphery of the range had lower levels of genetic diversity relative to the core. We suggest that the five main genetic groups delineated in our study warrant recognition as management units in terms of conservation planning. Intraregional population structure also points to the potential importance of other barriers to gene flow at finer spatial scales, although additional work is needed to better delineate these genetic groups.