The wood turtle (Glyptemys insculpta) is a freshwater species endemic to eastern North America and is currently listed as endangered by the International Union for Conservation of Nature. Wood turtle local populations are considered units for the species recovery, and are defined as discrete interbreeding populations in a distinct watershed; however, there are no studies to date supporting this definition. The main objective of this paper was to genetically characterize wood turtles from a northern portion of their range, and test an isolation-by-watershed hypothesis, the first of its kind at such a large geographical scale. Turtles were sampled in 24 watercourses from 12 different watersheds for a total of 331 individuals, each genotyped for nine microsatellite loci. Within each watershed, genetic diversity was similar between sites, and observed heterozygosity ranged from 0.677 to 0.754. Partial redundancy analyses then confirmed that watershed isolation contributed to 18% of the total observed genetic variation, while spatial autocorrelation and the post-glacial Expansion Model did not significantly explain any variation. Clustering methods revealed substantial spatial genetic structure, with sampled groups falling into ten nested hierarchical clusters. We recommend further consideration of these ten clusters to determine if they meet the evolutionary significance criterion to become designatable units, whose genetic structures were influenced by watershed structure.