We compared genetic differentiation among populations of the threatened massasauga rattlesnake (Sistrurus c. catenatus) using two types of nuclear molecular markers: randomly amplified polymorphic DNA (RAPD) markers and microsatellites. Analyses of molecular variance (AMOVA) and G(ST) and F(ST) analyses indicated that levels of among-population differentiation between regional populations (>100 km) were comparable for both markers. However, microsatellites were superior in population assignment tests and at discerning fine-scale genetic differentiation between subpopulations separated by tens of kilometers. These results argue that both types of markers are suitable for defining broad-scale genetic structures in snake populations and can provide important inputs into conservation initiatives of focal taxa. However, our analyses suggest that microsatellites 3re better for detecting structure at limited spatial scales.