The conservation and management of Blanding's Turtles (Emydoidea blandingii) has attracted considerable attention in recent years as the species is recognized as being at risk over much of its current range. We used seven variable microsatellites to examine five populations from Ontario (St. Lawrence River valley) to southeastern New York (Dutchess County) to assess genetic diversity and estimate gene flow, genetic drift, and the roles of distance and topography in dispersal of Blanding's Turtles. Three peripheral populations in the St. Lawrence River valley exhibited low levels of differentiation (FST < 0.07); past or current gene flow between these adjoining populations has likely been facilitated by the St. Lawrence River. A disjunct population in Dutchess County was divergent from other populations but showed no signs of loss of genetic diversity. A recently discovered population in Saratoga County displayed low levels of genetic diversity, possibly indicating that it has been isolated for some time. The St. Lawrence River valley populations appear to have sufficient habitat corridors to have maintained gene flow after the Wisconsin glaciation. This may not reflect current connectivity, however, and conservation measures should be aimed at maintaining connectivity and decreasing road mortality. Management plans for the Dutchess County population should consider maintaining connectivity among subpopulations a priority to avoid losing unique alleles to genetic drift. The Saratoga area should be investigated for other pockets of individuals that could potentially contribute to overall genetic diversity. The discontinuous distribution of this species within New York, coupled with regional differences in genetic variation, highlight the importance of maintaining all extant populations.