Eastern Diamondback Rattlesnakes (Crotalus adamanteus) are a species of conservation concern throughout their range. Yet, despite decades of population declines, C. adamanteus has not garnered protective status. With limited information on its general life history, more research is needed to manage C. adamanteus populations effectively, particularly at the southern extent of their distribution. To fill knowledge gaps in the ecology of C. adamanteus, we radiotracked six adult female and four male C. adamanteus from December 2015 to March 2018 (1,880 relocations) on a university campus in southwest Florida. Male snakes maintained large annual home ranges (mean = 65.7 ha 100% minimum complex polygons [MCP]) that were twice that of females (mean = 26.7 ha 100% MCP). Male snakes also made longer daily movements (mean = 39.4 ± confidence interval [CI] 34.7–44.0 m/d) than did females (mean = 16.7 ± CI 15.0–18.5 m/d), although both sexes showed variation among individuals. Snakes made considerable use of habitats adjacent to human development (<5 m from roadways and/or buildings), which consisted of more upland features than surrounding areas and may have provided thermoregulation benefits. However, snakes rarely crossed trafficked roads, which appeared to create barriers to their use of the landscape. Our research provides a better understanding of the spatial limits and dispersal patterns of C. adamanteus near the southernmost extent of its geographic range and within an urbanized landscape, which may assist in the implementation of effective management strategies.