Fire shapes habitats and therefore influences the genetic characteristics of populations. Florida scrub is a fire-dependent habitat with several precinctive species, including Florida Scrub Lizards (Sceloporus woodi). Fire history of scrub patches could affect the movement patterns of Florida Scrub Lizards, thereby altering the genetic characteristics of local populations. We characterized the effect of time since fire (TSF) on genetic diversity and differentiation at 6 microsatellite loci in the Florida Scrub Lizard (n = 413) collected from 17 sites in Highlands County, Florida. Private allelic richness was positively correlated with TSF (r = 0.56, P = 0.009). In sites with a TSF of 3–17 yr, TSF was negatively correlated with expected heterozygosity (r = −0.90, P = 0.009), inbreeding (r = −0.77, P = 0.04), allelic richness (r = −0.79, P = 0.03), and private allelic richness (r = −0.80, P = 0.03); TSF was positively correlated with mean pairwise relatedness (r = 0.85, P = 0.02). Therefore, a consequence of TSF is short-term change to local population genetics that is likely precipitated by responses of Florida Scrub Lizards to habitat modification. At a TSF of >20 yr, TSF and genetic diversity were not correlated, indicating that factors other than fire shape genetic diversity in long-unburned locations. We detected genetic differentiation using Bayesian clustering and estimates of F-statistics. Our results highlight the importance of consistent fire regimes in the Florida scrub on the genetic diversity of Florida Scrub Lizards. The presence of Florida Scrub Lizard populations in long-unburned sites, however, warrants further investigation.