Boreal Chorus Frogs (Pseudacris maculata) are widely distributed across North America; however, populations in parts of southeastern Canada and northeastern United States have been designated as either vulnerable or threatened because of human land use, and pressures from housing developments continue to jeopardize remnant populations found within fragmented habitats. A genetic characterization of Boreal Chorus Frogs sampled in Quebec and Ontario was conducted to help determine current levels of genetic variability, isolation, and to help identify important populations for conservation. The analysis involved 12 P. maculata-sampled locations using six microsatellite markers. Most exhibited discernible genetic differences, but some gene flow was still apparent among sites within and between three regions. We identified sampled locations with unique clustering results and little genetic similarity between neighboring sites. We propose that increased protection and monitoring of Boreal Chorus Frog populations, and establishing or expanding dispersal corridors among them, should be part of recovery planning.