Catadromous American eel ( Anguilla rostrata ) are native to Maine's Penobscot River watershed and historically have migrated through many of its tributaries prior to extensive damming. Recent restoration efforts, including dam removals, have improved connectivity in the lower reaches of the Penobscot River. Characterizing the extent of the American eel's distribution is important to inform restoration and identify extant barriers to migrations within the watershed. In the summer of 2023, we conducted eDNA surveys throughout the Penobscot River watershed to estimate the current distribution of the American eel and identify barriers to inland waters. Water samples were collected from 70 sites representing 37 rivers and streams; the presence or absence of American eel genetic markers within those samples was assessed using qPCR. We have shown that American eel are present in virtually the full extent of the area surveyed (68/70 sites). The results suggest that the majority of the main-stem dams may be passed by American eels at some level, with eel DNA being confirmed upstream of six dams. We confirmed the presence of American eels throughout the lower watershed with just 1 week of eDNA sampling and have highlighted this method for determining the species' access to habitat upstream of dams. The use of eDNA to sample locally (or regionally) for American eel may provide cost-effective information in data deficient areas and help assess the permeability of dam structures to diadromous species.