Studies about habitat fragmentation, in terms of how it affects gene flow and genetic variability, have traditionally been conducted on island-like systems in which the remaining habitats form patches embedded in a matrix. However, in agroecosystems, remaining habitats usually form linear strips along fence lines, roads, and water courses (“border” habitats). We used the rodent Calomys venustus, a species inhabiting borders in central Argentina agroecosystems, as a model to address how genetic variability is structured in linear habitats. A total of 359 rodents were captured seasonally from spring 2005 to winter 2006. Genetic variability at microsatellite loci was uniformly high, despite significant variation in population size during the sampling period. Genetic differentiation, spatial autocorrelation, and causal modeling analyses suggested that dispersion patterns in this species depend mainly on geographic distance, with unfavorable habitat like dirt roads and crop fields posing only weak (or no) resistance to dispersal. Small-scale spatial genetic structure was related to different space use patterns by females and males. Our results showed that, although greatly reduced in area, border habitats can support stable populations of species without loss of either variability or genetic connectivity.