Population genetic patterns can be affected by a number of factors, including life history characteristics and landscape features. Identifying general patterns of connectivity and key factors affecting these patterns is often central to effective ecological monitoring and management, particularly in areas with increasing urbanization. Examining patterns of connectivity and diversity of widespread and still abundant vertebrates, such as many cricetid rodents, can serve as a general indicator of habitat quality and connectivity. We investigated patterns of genetic diversity, differentiation, and population structure in 2 species of deer mice (Peromyscus californicus and P. fraterculus) occupying patches of native habitat separated by roadways in San Diego County, California. The 2 species are closely related and frequently co-occur at the same site. We collected samples from 5 widely distributed sites throughout the county and analyzed them using 10–12 microsatellites. We found similar levels of genetic diversity in both species, with allelic richness ranging from 4.47 to 8.81 and observed heterozygosity values from 0.60 to 0.79. In general, we found that P. californicus exhibits a pattern of isolation by distance at a finer spatial scale than does P. fraterculus. Populations of both species that were separated by major roads were not strongly genetically differentiated, with one exception: at 1 site, P. fraterculus exhibited signs of minor differentiation across a roadway. However, subsequent landscape modeling at this site was unsuccessful in detecting a correlation between specific landscape features and genetic differentiation. In general, our study shows that small rodent populations may be robust with respect to impacts of recent anthropogenic fragmentation by roadways.