Roads can fragment animal populations by reducing gene flow, which can lead to drift and the loss of genetic diversity. One of the principle signatures of reduced gene flow is increased genetic differentiation in isolated populations, and evidence that roads contribute to such differentiation has been reported for several species. We used microsatellites to examine whether six roads led to increased genetic differentiation in red-backed salamanders (Plethodon cinereus). These six roads included one divided interstate highway, one undivided four-lane highway, and four secondary roads. We found that the genetic distance between plots that were bisected by the interstate highway was significantly greater than the genetic distance between equidistant plots on the same side of the highway. However, for the five smaller roads, plots across the road were no more genetically distinct than were plots on the same side of the road. Bayesian clustering methods also supported both of these findings. The optimal clustering of plots for the interstate highway consisted of two clusters that corresponded to the two sides of highway. For the other five sites, the optimal grouping consisted of a single cluster containing all of the plots. Our findings suggest that gene flow across very large roads is rare and that bisected red-backed salamander populations are likely to diverge from one another. For smaller roads, our results imply that the indirect effects of roads on genetic population structure are probably less of a pressing concern for terrestrial salamanders than are the direct effects of mortality and habitat alteration.