AbstractObjectiveSea Lamprey Petromyzon marinus provide important ecological services within their native range, such as nutrient cycling, and can also act as a prey source for other species. Adult Sea Lamprey must access freshwater rivers to spawn, and because of this they are susceptible to changes in river connectivity. Human‐made structures, such as dams, can exclude them from usable habitat. Sea Lamprey dam passage has not been extensively studied in Maine, despite Maine being within the native range of this species. The goals of this study were to evaluate upstream passage efficiency at the Milford Dam on the Penobscot River, Maine, and to provide comprehensive information about adult Sea Lamprey passage at five other dams throughout the Penobscot River watershed.MethodsIn 2020–2021 we captured and tagged 150 Sea Lamprey at the Milford Dam, the lowest dam in the Penobscot River, Maine, and displaced them downstream to assess passage efficiency at this dam and five upstream dams. In 2020, 50 Sea Lamprey were released on the east shore of the river downstream of Milford Dam; in 2021, the east shore release was repeated with an additional 50 fish and another 50 fish were released on the west shore.ResultBetween 70–82% of Sea Lamprey were observed passing Milford Dam again after mean delay times of 9–11 days. The release location did not affect dam passage success or the amount of time that was required to locate and use the passage structures. Sea Lampreys from both release groups were equally likely to approach the entrance to the fishway upon returning to Milford Dam, despite the fishway being located against the eastern shore of the river. However, high flows shortly after release may have resulted in higher attraction to the fishway in 2020. Passage success at dams upstream of Milford was highly variable. All Sea Lamprey were able to successfully navigate past West Enfield Dam (100% passage, n = 63), whereas Brownsmill Dam apparently acted as a complete barrier to further migration (0% passage, n = 7). Fish from all years and release groups together had a median upstream migration distance of 38.8 km after fish had passed Milford Dam, and a maximum observed upstream travel distance of approximately 100 km, indicating that most tagged Sea Lamprey ended their migration in the vicinity of a dam.ConclusionThe results of this study indicate that Sea Lamprey have high passage efficiency at the Milford Dam and highlight areas within the Penobscot River basin—such as the Brownsmill Dam—where passage facilities are currently inadequate for Sea Lamprey.