The Penobscot River Restoration Project in Maine was a large river rehabilitation project that culminated in the removal of the two lowermost dams and improvements to fish passage on several remaining dams. Fish assemblages were surveyed for 3 years prior to rehabilitation, 3 years after rehabilitation, and 8 years after rehabilitation. Approximately 475 km of shoreline were sampled via boat electrofishing, yielding 133,394 individual fish of 41 species. The greatest shifts in assemblage structure occurred immediately after dam removal in formerly impounded sections, with an increased prevalence of riverine and migratory species. Long‐term sampling documented changes within tributaries and tidally influenced river segments, where large schools of adult and young‐of‐the‐year alosines increased in abundance. Upstream of the lowermost dam, the river remains dominated by lacustrine species, while adult anadromous fishes continue to be most abundant immediately downstream of the lowermost dam. Our results provide increased evidence that dam removals result in altered fish assemblages, which are now dominated by riverine and anadromous species in previously impounded habitats. Alosines in the Penobscot River have exhibited the greatest long‐term response to river restoration efforts.