For polytypic mammal species, biogeographic barriers including rivers have often been used to delineate taxonomic units under the assumption that barriers have structured their distribution. We tested the importance of major riverine systems as biogeographic barriers in fox squirrels (Sciurus niger) across the state of Florida, where 4 currently recognized subspecies are delineated at major rivers. We also explored whether phylogeographic structure may be limited to ecologically divergent subspecies, specifically between S. n. avicennia and S. n. shermani. Using a multilocus approach to examine diversity, we found that restricted gene flow was only present between S. n. avicennia, located south of the Caloosahatchee River in south Florida, and the rest of S. niger, which is widespread across the state. Mitochondrial DNA revealed that 2 divergent groups of haplotypes are present and widespread across Florida, thus supporting the hypothesis that fox squirrels persisted in multiple refugia during the Pleistocene, and that S. n. avicennia diverged ecologically from other populations of fox squirrels relatively recently. This was supported by isolation with migration models that indicated matrilineal isolation since the onset of divergence between S. n. avicennia and S. n. shermani, which corresponds to the onset of ecological divergence in south Florida during the early Holocene. Isolation by distance at 8 microsatellite loci from the western panhandle to the southern end of the peninsula was significant only when S. n. avicennia was included; however, this was due to the hierarchical genetic patterns identified between S. n. avicennia and the other subspecies as determined by Bayesian clustering, and not due to spatially restricted dispersal. We postulate that the demographic isolation of S. n. avicennia is the result of adaptation to the unique ecological conditions of south Florida.