Agricultural ecosystems are spatially and temporally dynamic systems that support wildlife populations in addition to food production for humans. The composition and configuration of land cover within agricultural systems, which often varies seasonally or annually, can have profound impacts on biodiversity. Specifically, landscape structure can influence animal dispersal and distribution. Depending on species-specific ecology, dispersal may be restricted, resulting in smaller, reproductively isolated populations. Few studies have characterized the spatial genetic structure of small mammal species in agricultural landscapes, despite their importance to crop pest management and food webs within agroecosystems. We characterized the spatial genetic structure of 2 co-distributed Peromyscus species (Peromyscus leucopus and Peromyscus maniculatus bairdii), and quantified relationships between spatial genetic structure based on interindividual relatedness resolved from 10 microsatellite loci, and landscape features hypothesized to influence dispersal. We found significant spatial autocorrelation in interindividual relatedness over interindividual distances of 1,800 m for P. leucopus and 300 m for P. maniculatus bairdii. We also identified distinct genetic clusters of interbreeding individuals for both species. Spatial genetic structure of P. leucopus was significantly related to the distribution of roads, indicating that highly fragmented landscapes could negatively affect dispersal and gene flow. Given the large and growing footprint of agriculture globally, it is important that studies of the effects that landscape features have on dispersal of wildlife species include human-dominated landscapes.