Topographical features and environmental variables form geographic range boundaries and limit species' distributions. Studies of Wood Turtle (Glyptemys insculpta) limitations in the northern reaches of their geographic range are fairly well documented; however, few studies exist that investigate ecological factors that affect distribution at the southern extent of their range. Our objectives were to 1) determine aquatic and terrestrial habitat characteristics relevant to Wood Turtle presence or absence and 2) determine the approximate southern geographic boundary of Wood Turtles in the Cacapon River Watershed, West Virginia, USA. We conducted random surveys of 100 sites along the Lost and North rivers during summer 2010, and 64% contained Wood Turtles. Proximity to the Cacapon River, elevation, soil pH, canopy cover, and slope influenced the presence of Wood Turtles. In addition, Wood Turtles occurred at sites with higher herbaceous species richness and diversity, especially in locations along the North River. Vegetative community composition differed between terrestrial sites associated with these two rivers in the field and shrub layers (regardless of turtle presence), and differed in the site × river interaction in the tree layer. Wood Turtles on the Lost and North rivers, at their southern range limits in West Virginia, are associated with lower elevation, gentler slopes, higher soil pH, and higher tree canopy cover, and also are located closer to the confluence of the Cacapon River. We recommend creating and managing riparian buffers along waterways where Wood Turtles occur to provide essential terrestrial habitat and to promote adult survivorship and population stability.