This study evaluated Atlantic salmon Salmo salar smolt survival through the lower Penobscot River, Maine, U.S.A., and characterized relative differences in proportional use and survival through the main-stem of the river and an alternative migration route, the Stillwater Branch. The work was conducted prior to removal of two main-stem dams and operational changes in hydropower facilities in the Stillwater Branch. Survival and proportional use of migration routes in the lower Penobscot were estimated from multistate (MS) models based on 6 years of acoustic telemetry data from 1669 smolts and 2 years of radio-telemetry data from 190 fish. A small proportion (0·12, 95% c.i. = 0·06–0·25) of smolts used the Stillwater Branch, and mean survival through the two operational dams in this part of the river was relatively high (1·00 and 0·97). Survival at Milford Dam, the dam that will remain in the main-stem of the Penobscot River, was relatively low (0·91), whereas survival through two dams that were removed was relatively high (0·99 and 0·98). Smolt survival could decrease in the Stillwater Branch with the addition of two new powerhouses while continuing to meet fish passage standards. The effects of removing two dams in the main-stem are expected to be negligible for smolt survival based on high survival observed from 2005 to 2012 at those locations. Survival through Milford Dam was been well below current regulatory standards, and thus improvement of passage at this location offers the best opportunity for improving overall smolt survival in the lower river.