Identifying how natural (i.e., unaltered by human activity) and anthropogenic landscape variables influence contemporary functional connectivity in terrestrial organisms can elucidate the genetic consequences of environmental change. We examine population genetic structure and functional connectivity among populations of a declining species, the Blainville’s horned lizard (Phrynosoma blainvillii), in the urbanized landscape of the Greater Los Angeles Area in Southern California, USA. Using single nucleotide polymorphism data, we assessed genetic structure among populations occurring at the interface of two abutting evolutionary lineages, and at a fine scale among habitat fragments within the heavily urbanized area. Based on the ecology of P. blainvillii, we predicted which environmental variables influence population structure and gene flow and used gravity models to distinguish among hypotheses to best explain population connectivity. Our results show evidence of admixture between two evolutionary lineages and strong population genetic structure across small habitat fragments. We also show that topography, microclimate, and soil and vegetation types are important predictors of functional connectivity, and that anthropogenic disturbance, including recent fire history and urban development, are key factors impacting contemporary population dynamics. Examining how natural and anthropogenic sources of landscape variation affect contemporary population genetics is critical to understanding how to best manage sensitive species in a rapidly changing landscape.