Roads can act as significant barriers to wildlife dispersal, creating small fragmented populations at increased risk for genetic diversity loss and inbreeding. The magnitude of road impacts on wildlife populations depends on road characteristics and the species considered; therefore it is difficult to generalize results among systems. To begin to understand how Northern California highways affect native mammals, we determine whether SR 50 and I-80 in the Sierra Nevada and I-680 and I-580 in the Bay Area act as barriers to coyote dispersal and gene flow. We collected samples from road killed coyotes and non-invasive sources (hair and scat) on either side of each highway and used landscape genetic analyses to determine how genetic diversity is partitioned across these putative barriers. Population assignment tests infer the number of populations and identify migrants dispersing across highways. Spatial autocorrelation analyses determine whether genotypes are distributed randomly in the landscape or are restricted by highway barriers. Our study reveals significant patterns of coyote population subdivision on either side of major Northern California highways. Our results will assist agencies in developing mitigation plans to reduce the impact of transportation on native wildlife population.