19Apex predators are important indicators of intact natural ecosystems. They are also sensitive to 20 urbanization because they require broad home ranges and extensive contiguous habitat to support their 21 prey base. Pumas (Puma concolor) can persist near human developed areas, but urbanization may be 22 detrimental to their movement ecology, population structure, and genetic diversity. To investigate 23 potential effects of urbanization in population connectivity of pumas, we performed a landscape genomics 24 study of 134 pumas on the rural Western Slope and more urbanized Front Range of Colorado, USA. Over 25 12,000 single nucleotide polymorphisms were genotyped using double-digest, restriction site-associated 26 DNA sequencing (ddRADseq). We investigated patterns of gene flow and genetic diversity, and tested for 27 correlations between key landscape variables and genetic distance to assess the effects of urbanization and 28 other landscape factors on gene flow. Levels of genetic diversity were similar for the Western Slope and 29 Front Range, but effective population sizes were smaller, genetic distances were higher, and there was 30 more overall population substructure in the more urbanized Front Range. Forest cover was strongly 31 positively associated with puma gene flow on the Western Slope, while impervious surfaces restricted 32 gene flow and more open, natural habitats enhanced gene flow on the Front Range. Landscape genomic 33 analyses revealed differences in puma movement and gene flow patterns in rural versus urban settings. 34 Our results highlight the utility of dense, genome-scale markers to document subtle impacts of 35 urbanization on a wide-ranging carnivore living near a large urban center.36 37