The Penobscot River drains the largest watershed in Maine and once provided spawning and rearing habitats to 11 species of diadromous fishes. The construction of dams blocked migrations of these fishes and likely changed the structure and function of fish assemblages throughout the river. The proposed removal of two main-stem dams, improved upstream fish passage at a third dam, and construction of a fish bypass on a dam obstructing a major tributary is anticipated to increase passage of and improve habitat connectivity for both diadromous and resident fishes. We captured 61 837 fish of 35 species in the Penobscot River and major tributaries, through 114 km of boat electrofishing. Patterns of fish assemblage structure did not change considerably during our sampling; relatively few species contributed to seasonal and annual variability within the main-stem river, including smallmouth bass Micropterus dolomieu, white sucker Catostomus commersonii, pumpkinseed Lepomis gibbosus, and golden shiner Notemigonus crysoleucas. However, distinct fish assemblages were present among river sections bounded by dams. Many diadromous species were restricted to tidal waters downriver of the Veazie Dam; Fundulus species were also abundant within the tidal river section. Smallmouth bass and pumpkinseed were most prevalent within the Veazie Dam impoundment and the free-flowing river section immediately upriver, suggesting the importance of both types of habitat that supports multiple life stages of these species. Further upriver, brown bullhead Ameiurus nebulosus, yellow perch Perca flavescens, chain pickerel Esox niger, and cyprinid species were more prevalent than within any other river section. Our findings describe baseline spatial patterns of fish assemblages in the Penobscot River in relation to dams with which to compare assessments after dam removal occurs.