Throughout its distribution in North America, the threatened eastern massasauga rattlesnake (Sistrurus c. catenatus) persists in a series of habitat-isolated disjunct populations of varying size. Here, we use six microsatellite DNA loci to generate information on the degree of genetic differentiation between, and the levels of inbreeding within populations to understand how evolutionary processes operate in these populations and aid the development of conservation plans for this species. Samples were collected from 199 individuals from five populations in Ontario, New York and Ohio. Our results show that all sampled populations: (i) differ significantly in allele frequencies even though some populations are < 50 km apart, and may contain genetically distinct subpopulations < 2 km apart; (ii) have an average of 23% of alleles that are population specific; and (iii) have significant FIS values (mean overall FIS = 0.194) probably due to a combination of Wahlund effects resulting from fine-scale genetic differentiation within populations and the presence of null alleles. Our results imply that massasauga populations may be genetically structured on an extremely fine scale even within continuous populations, possibly due to limited dispersal. Additional information is needed to determine if dispersal and mating behaviour within populations can account for this structure and whether the observed differentiation is due to random processes such as drift or to local adaptation. From a conservation perspective, our results imply that these massasauga populations should be managed as demographically independent units and that each has high conservation value in terms of containing unique genetic variation.