We report a survey of microsatellite DNA variation in Atlantic salmon from the unimpounded lower reaches of Maine's Penobscot River. Our analysis indicates that Atlantic salmon in the Penobscot River are distinct from other populations that have little or no history of human-mediated repopulation, including two of its tributaries, Cove Brook and Kenduskeag Stream, another Maine river, the Ducktrap, and Canada's Miramichi and Gander rivers. Significant heterogeneity was detected in allele frequency among all three subpopulations sampled in the Penobscot drainage. The high resolution of the 12-locussuite was quantified using maximum likelihood assignment tests, which correctly identified the source of 90.4–96.1% of individuals from within the Penobscot drainage. Current populations are clearly isolated from each other, however we are unable to determine from the present data whether the populations in Cove Brook and Kenduskeag Stream are recently diverged from populations stocked into the Penobscot River over the last century, or are aboriginal in origin. The degree of population structure identified in the Penobscot drainage is noteworthy in light of its lengthy history of systematic restocking, the geographic proximity of the subpopulations, and the extent of the differentiation. Similar population structure on this extremely limited geographic scale could exist among Atlantic salmon runs elsewhere in Maine and throughout the species' range and should be taken into account for future management decisions.