Genetic differentiation among populations of the moor frog (Rana arvalis) was tested on a spatial scale where some dispersal between populations is expected to occur, in a landscape in The Netherlands that has become fragmented fairly recently, in the 1930s. Five microsatellite loci were used, with 2–8 alleles per locus. FIS was 0.049 across loci, and most populations were in HW equilibrium. The degree of population subdivision was low (FST=0.052). A significant positive correlation between genetic distance and geographical distance was found, indicating a limitation in dispersal among populations due to distance. To test the impact of the landscape mosaic on the connectivity between patches, distance measures were corrected for relative amounts of habitat types with known positive or negative influence on moor frog dispersal. Notably, the resistance variable for the fraction of negative linear elements (roads and railways) gave a higher explanatory value than geographical distance itself. Therefore, it is particularly the number of barriers (roads and railways) between populations that emerges as a factor that reduces exchange between populations. It is concluded that genetic techniques show promise in determining the influence of landscape connectivity on animal dispersal.