We compared the efficiency of stratified random and fixed-station sampling designs to characterize fish assemblages in anticipation of dam removal on the Penobscot River, the largest river in Maine. We used boat electrofishing methods in both sampling designs. Multiple 500-m transects were selected randomly and electrofished in each of nine strata within the stratified random sampling design. Within the fixed-station design, up to 11 transects (1,000 m) were electrofished, all of which had been sampled previously. In total, 88 km of shoreline were electrofished during summer and fall in 2010 and 2011, and 45,874 individuals of 34 fish species were captured. Species-accumulation and dissimilarity curve analyses indicated that all sampling effort, other than fall 2011 under the fixed-station design, provided repeatable estimates of total species richness and proportional abundances. Overall, our sampling designs were similar in precision and efficiency for sampling fish assemblages. The fixed-station design was negatively biased for estimating the abundance of species such as Common Shiner Luxilus cornutus and Fallfish Semotilus corporalis and was positively biased for estimating biomass for species such as White Sucker Catostomus commersonii and Atlantic Salmon Salmo salar. However, we found no significant differences between the designs for proportional catch and biomass per unit effort, except in fall 2011. The difference observed in fall 2011 was due to limitations on the number and location of fixed sites that could be sampled, rather than an inherent bias within the design. Given the results from sampling in the Penobscot River, application of the stratified random design is preferable to the fixed-station design due to less potential for bias caused by varying sampling effort, such as what occurred in the fall 2011 fixed-station sample or due to purposeful site selection.