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The American Eel Anguilla rostratahistorically was one of the most common fish species in Atlantic coast watersheds, but extensive dam construction and other factors caused a widespread population decline. One of the watersheds where American Eels have declined considerably is the Mohawk River in eastern and central New York. Recent attempts to characterize the distribution and abundance of American Eels in this watershed have been ineffective, and the extent to which a series of locks and dams on the Hudson River and lower Mohawk River limits use of the watershed is unclear. We developed a model between environmental DNA (eDNA) quantity and American Eel abundance in the Hudson River watershed in which the DNA concentration in water samples explained up to 65% of the variability in eel density and 56% of the variability in eel biomass. We then used this relationship to interpret eDNA data collected twice from 36 sites across the Mohawk River watershed in 2021 and make inferences about the distribution and abundance of American Eels. American Eel DNA was detected almost exclusively in the downstream‐most 4 km of the Mohawk River within a series of barriers. The concentration of DNA was reduced by approximately 80% across each successive upstream barrier before becoming too low to detect consistently. Our data suggest that eel population density was high in the Hudson River estuary and declined rapidly in the lower Mohawk River, and the species was nearly absent or undetectable in the Mohawk River and its tributaries upstream of the Crescent Dam and the Waterford Flight of Locks. Barriers appear to be largely restricting American Eels from using over 99% of the Mohawk River watershed. Therefore, improvements in fish passage at dams and hydroelectric facilities in the region could help the American Eel to regain access to this part of its native range. Impact statementThis work developed and utilized a novel approach for estimating the distribution and abundance of an imperiled migratory fish species, American Eel, which can be used to identify barriers to upstream movement and inform conservation efforts. This work developed and utilized a novel approach for estimating the distribution and abundance of an imperiled migratory fish species, American Eel, which can be used to identify barriers to upstream movement and inform conservation efforts.