The fragmentation of natural habitat is a major threat to the conservation of species because it reduces genetic variability and increases population extinction risk. The leading causes of fragmentation in the Ocala National Forest (ONF) are clearcut logging for wood pulp and roads (e.g., Florida State Route 40 or FSR-40). We used five microsatellite (Msat) loci and 301 base pairs of mitochondrial DNA (mtDNA) to estimate genetic differentiation among subpopulations of Florida Scrub Lizards (Sceloporus woodi) inhabiting “islands” of suitable habitat across the ONF. Pairwise comparisons of subpopulations revealed that 52.4% of FST values were different for mtDNA and 71.4% for Msats, including differentiation over short distances (2 km) between subpopulations separated by FSR-40. A pattern of isolation-by-distance (IBD) was detected with mtDNA; however, there was no IBD evident in the Msat data. Analysis of molecular variance (AMOVA) revealed that grouping subpopulations based on their position to FSR-40 (north/south) only explained 0.02% and 3.30% of the among-group variation for Msats and mtDNA, respectively. An additional AMOVA (mtDNA) grouping by habitat type (longleaf pine vs. sand–pine scrub) increased the among-group variation to 22.8%. The results reveal a pattern of genetic patchiness in a terrestrial landscape that is anthropogenically fragmented. We hypothesize that logging, roads, and habitat variation might all have consequences for the population dynamics and genetic diversity of S. woodi on an ecological time scale.