Lake Sturgeon (Acipenser fulvescens) are large, long-lived fish, characteristics conducive to the bioaccumulation of mercury, a harmful contaminant. This study measured total mercury (THg) in the axial musculature of 319 Lake Sturgeon captured between 2002 and 2022 from six river reaches on the Winnipeg, Nelson, Churchill, and Hayes rivers in the province of Manitoba, Canada. Since the 1900s, all sampled rivers except the Hayes were impacted by hydroelectric development, which can increase THg concentrations in fish for several decades following inundation. Concentrations of THg in Lake Sturgeon measuring 171-1,435 mm fork length ranged from less than the detection limit (0.005 ppm) to 0.698 ppm wet weight. THg concentrations increased significantly with fish length for all waterbodies analyzed except for the Hayes River where the sample size and length range were small. Mean length-standardized THg concentrations ranged from 0.069 to 0.177 ppm and differed significantly between waterbodies. Concentrations were lowest where the majority of samples were collected from fish that had been hatchery-reared and stocked. A paucity of historical data precludes the isolation of effects of hydroelectric impoundment on THg levels in Lake Sturgeon, but concentrations are not particularly high in any of the waterbodies examined herein. Of the 35 Lake Sturgeon also analyzed for diet composition, 34 had identifiable gastrointestinal contents. Ten different prey taxa were identified, including fish. Chironomids dominated dietary numerical abundance. The number of taxa increased with fish length to include larger, and therefore potentially more THg-rich organisms.