A species of conservation concern, the Flat-tailed Horned Lizard (Phrynosoma mcallii; FTHL) has been petitioned for listing under the US Endangered Species Act four times. Although management and monitoring strategies have been developed to help prevent the need for federal listing, genetic information about the species is limited. Previous genomic research has focused on populations across the range, but small sample sizes have limited resolution for detecting fine-scale population structure and barriers to gene flow. We genotyped over 15,000 SNPs using double-digest restriction site-associated DNA sequencing to assess population structure, potential barriers to gene flow, trends in genetic variation over time, and the effect of geography on distribution of genetic variation in 175 FTHL from the Yuma Desert Management Area. No major changes in genetic variation or inbreeding occurred between samples from 2000 and 2019. Our analyses also provide support for a single, nearly panmictic population in the management area; however, we detected fine-scale genetic structure, which may be explained by the presence of highways that appear to subdivide populations separated by relatively short geographic distances. Although low levels of inbreeding and genetic differentiation among sites suggest fragmentation by highways is not yet a pressing threat to population viability, future management strategies focused on safe passage of individuals across heavily trafficked highways may help slow loss of connectivity. These results highlight the importance of continued genomic monitoring of populations to better detect long-term trends in genetic variability and effects of barriers to gene flow.