In an urbanizing region of Southwestern Ontario, a declining population of Eastern Massasauga Rattlesnakes ('Sistrurus catenatus catenatus') persists in taligrass prairie remnants. Maintaining an adequate level of landscape connectivity is a recommended strategy to encourage dispersal between habitat patches and to reduce the extinction risk of this genetically and ecologically distinct population. The goal of this study was to identify potential connectivity pathways between habitat patches for this species by using a GIS least-cost model and to evaluate the outputs with road mortality data. Results identified seven pathways between five core habitat patches which were validated with aerial imagery and mortality data. A subset of pathways requires further study. This research will guide the location of interventions aimed at increasing connectivity for this species, provide a basis for connectivity design for other species within the study landscape, and inform the use of a modelling approach to connectivity analysis in urban landscapes.