While many studies have examined the barrier effects of large rivers on animal dispersal and gene flow, few studies have considered the barrier effects of small streams. We used displacement experiments and analyses of genetic population structure to examine the effects of first-order and second-order streams on the dispersal of terrestrial red-backed salamanders, Plethodon cinereus (Green, 1818). We marked red-backed salamanders from near the edges of one first-order stream and one second-order stream, and experimentally displaced them either across the stream or an equal distance farther into the forest. A comparison of return rates indicated that both streams were partial barriers to salamander movement, reducing return rates by approximately 50%. Analysis of six microsatellite loci from paired plots on the same side and on opposite sides of the second-order stream suggested that the stream did contribute to genetic differentiation of salamander populations. Collectively, our results imply that low-order streams do influence patterns of movement and gene flow in red-backed salamanders. We suggest that given the high density of first-order and second-order streams in most landscapes, these features may have important effects on species that, like red-backed salamanders, have limited dispersal and large geographic ranges.