The genetic structuring of populations can be influenced by present processes and past events. One of the largest historical events to affect the distribution and genetic characteristics of present-day North American biota is the Pleistocene glaciation. Thus, the study of post-glacial colonization patterns of species in northern landscapes can relay important ecological information, as species had to expand their range extensively following the retreat of the glaciers and are often at the terminal end of their expansion. These species consequently exhibit the genetic fingerprints of sequential founder events, in turn decreasing the genetic variation available for adaptation. Using amphibians to investigate post-glacial range expansion is advantageous, as they have limited dispersal abilities revealing fine-scale patterns and they are thought to be one of the first vertebrates to colonize post-glacial habitat. Therefore, to model the phylogeography of a primary colonizer and the population structure of anurans in northern landscapes, population genetics analyses of wood frogs (Lithobates sylvaticus) were performed in the James Bay area. Wood frogs were sampled from 17 localities around James Bay and genetic analyses were conducted with seven microsatellite loci and mitochondrial DNA sequences of the ND2/tRNATRP genes. Results show that the post-glacial recolonization of the James Bay area by wood frogs originated from the putative refugium in western Wisconsin, an area known as the Driftless Area. Two routes were taken by founders to colonize the James Bay area: one north-west of Lake Superior, colonizing western Ontario, and one through the Upper Peninsula of Michigan, colonizing southern and eastern Ontario and western Québec. Interestingly, the meeting of the two lineages south-west of James Bay led to the establishment of a zone of higher genetic variation than expected under the founder effect hypothesis. Additionally, population structure analyses revealed the segregation of three genetic populations east, north-west, and south-west of the bay, the latter showing the highest genetic variation and likely representing a zone of secondary contact. This study shows that past events such as post-glacial range expansions can explain present patterns of genetic variation and population structure, and that studies in northern landscapes may be very useful in understanding genetic patterns throughout the range of a species.