Roads and highways contribute enormously to habitat fragmentation, because they can inhibit or even block animal movement across them, which may result in the ultimate division of the populations adjacent to the roads into smaller isolated subpopulations. The isolation reduces gene flow and increases risk of extinction due to a decrease in the genetic diversity of the isolated population. The aim of the present study is to determine whether highways can cause genetic subdivision of the bank vole Myodes glareolus (Schreber, 1780) and yellow-necked mouse Apodemus flavicollis (Melchior, 1834). The study was carried out at three sites in the Highway D1 (Prague-Brno) in the Czech Republic, where a previous study demonstrated a barrier effect of the highway avoiding the interchange of individuals of both species. The genetic structure was determined from the analysis of six DNA microsatellites loci in M. glareolus and five in A. flavicollis. We found only weak genetic differences between populations living at opposite sides of the highway in either of the species and a low degree of subdivision, but significant positive correlation between genetic and geographical distance, which suggests isolation by distance in both species.