Movement behavior is a critical process that interacts with landscape structure to affect population connectivity and persistence in fragmented or altered landscapes. The purpose of our study was to test whether different substrates (forest litter, soil, grass, gravel, and asphalt) found in fragmented forested landscapes affected the movement behavior of the Southern Graycheek Salamander (Plethodon metcalfi Brimley, 1912). Latency period of the salamanders was highest on grass substrate and significantly lower only on soil substrate. Sinuosity of the movement path of salamanders was lowest and contained more turns in grass and was significantly higher than only gravel and asphalt substrates. Velocity of the salamanders was highest on asphalt substrate but was not different from gravel substrate. Velocity was higher on asphalt than on grass, forest, or soil, and velocity was higher on gravel than on grass substrate. The results indicated that P. metcalfi reacted differently to natural and anthropogenic substrates, and we suggest that these behavioral differences could have both positive and negative implications for movement success and habitat resistance in forested landscapes fragmented by roads and development.