The Florida scrub lizard, Sceloporus woodi, is endemic to scrub habitat patches along the central portion of the Florida peninsula and xeric coastal regions. Scrub ecosystems are the patchily distributed remnants of previously widespread habitats formed during the Pleiocene and early Pleistocene. Scrub lizards appear to have limited dispersal capabilities due to high habitat specificity and low mobility. To assess the population structure and phylogeography of S. woodi, 135 samples were collected from 16 patches on five major ridges in Florida, USA. Analysis of 273 bp of mitochondrial DNA (mtDNA) cytochrome b reveals a very strong geographic distribution of genetic diversity. Haplotype frequencies are significantly different in 63 of 66 comparisons between patches. With one exception, samples from the five major ridges are characterized by fixed differences in haplotype distribution and deep evolutionary separations (3–10%). Fixed genetic differences were also observed between northern and southern segments of several ridges. Analysis of molecular variance (amova) shows an estimated 10.4% total genetic variation within patches, 17.5% among patches (within ridges), and 72.1% among ridges. This strong population structure among patches within ridges indicates that the distribution of S. woodi is tightly linked to sandy scrub habitat and that the discontinuous distribution of scrub habitats significantly inhibits dispersal and gene flow. Phylogeographic analyses indicate a pattern of dispersal down the Florida peninsula during the late Pliocene–early Pleistocene, followed by habitat fragmentation and vicariant isolation events. Therefore, the deep genetic structuring among scrub lizard populations on separate ridges is attributed to ancient isolation events induced by a shift from dry (xeric) to wet (mesic) conditions on the Florida peninsula. These findings indicate that some scrub lizard populations have persisted in isolation for time frames in excess of 1 Myr, providing a case history on the genetic consequences of habitat fragmentation.