For organisms that reproduce in discrete habitat patches, land cover between patches (known as the matrix) is important for dispersal among breeding sites. Models of patchy populations often incorporate information on the permeability of the matrix to dispersal, sometimes based on expert opinion. I estimated the relative resistance to gene flow of land cover types and barriers using FST calculated from microsatellite markers in two amphibians, within an 800-km2 area in northern Switzerland. The species included a frog (Rana temporaria: 996 individuals, 48 populations, seven markers) and a newt (Triturus alpestris: 816 individuals, 41 populations, seven markers). Open fields and urban areas were more resistant to gene flow than forested land; roads and highways also reduced permeability. Results were similar for the two species. However, differences in resistance among matrix elements were relatively low: gene flow through urban areas was reduced by only 24–42% relative to forest; a divided highway reduced gene flow by 11–40% and was 7–8 times more resistant than a secondary road. These data offer an empirically based alternative to expert opinion for setting relative resistance values in landscape models.