Sceloporus woodi (Florida Scrub Lizard) offers an excellent opportunity to contrast connected and fragmented habitats to determine how historical patterns influence genetic differentiation in fragmented habitats. We screened six microsatellite loci in S. woodi (N = 154) from seven sites at Archbold Biological Station, a large, connected scrub habitat where there is a potential for gene flow in spite of the low dispersal in S. woodi. We compared genetic differentiation of S. woodi in the connected scrub to that previously detected among a cluster of naturally fragmented scrub patches. We also performed simulations to determine how fragmentation would alter genetic differentiation in the connected scrub. Sceloporus woodi had modest, fine-scale genetic differentiation that followed isolation-by-distance and habitat structure in the connected scrub. There was less differentiation in the connected scrub than observed among the naturally fragmented scrub patches. Spatially explicit Bayesian clustering with TESS and BAPS identified two groups among the seven sites and θ_ST estimates revealed significant differentiation among sites (θ_ST = 0.052; P < 0.001). A positive relationship occurred between geographic distance and genetic differentiation (r = 0.226; P = 0.04). Limited dispersal likely has generated isolation-by-distance with a gradient of genetic differentiation among sites. When the geographic relationship among sites was considered, simulations indicated that fragmentation would tend to increase genetic differentiation immediately after fragmentation, yet fragmentation reduced genetic differentiation in some cases. Thus, historical patterns of genetic differentiation may play an important role in determining the population structure of a species after habitat fragmentation.