Understanding interactions between roadways and population structure and movements of wildlife is key to mitigating “road effects” associated with increasing urbanization of the landscape. Aquatic turtles are a useful focal group because (1) population persistence is sensitive to mortality of individuals upon roads; (2) turtles frequently move among wetlands and encounter roads, and (3) turtles are an important component of vertebrate biomass in aquatic ecosystems. From 2005 to 2007, we examined the effects of urbanization on local- and landscape-scale populations of turtles. To do so, we sampled and marked turtles in 15 ponds arranged along a steep, urban–rural gradient in central New York State. We captured 494 turtles, representing 327 individuals, the majority of which were common snapping turtles Chelydra serpentina (n = 191) and eastern painted turtles Chrysemys picta picta (n = 122). At the local population (pond) scale, a higher proportion of female snapping turtles in ponds was associated with lower road densities within 500 m of ponds. The mean size of both species of turtle increased in ponds with a lower density of roads within 100 m. At the landscape-level, we observed fewer turtles dispersing through urbanized habitat than forested, and fewer movements through areas with a higher density of roads. Our study suggests that roads alter both local- and landscape-level turtle populations through a loss of female turtles, and by reducing movement between ponds. By extension, the study targets key landscape features upon which to focus mitigation efforts.