Tracking the uptake and transfer of toxic chemicals, such as mercury (Hg), in aquatic systems is challenging when many top predators are highly mobile and may therefore be exposed to chemicals in areas other than their location of capture, confounding interpretation of bioaccumulation trends. Here we show how the application of a less commonly used ecological tracer, stable sulfur isotope ratios (34S/32S, or δ34S), in a large river-delta-lake complex in northern Canada allows differentiation of resident from migrant fishes, beyond what was possible with more conventional 13C/12C and 15N/14N measurements. Though all large fishes (n = 105) were captured in the river, the majority (76%) had δ34S values that were indicative of the fish having been reared in the lake. These migrant fishes were connected to a food chain with greater Hg trophic magnification relative to the resident fish of the river and delta. Yet, despite a shallower overall trophic magnification slope, large river-resident fish had higher Hg concentrations owing to a greater biomagnification of Hg between small and large fishes. These findings reveal how S isotopes can trace fish feeding habitats in large freshwater systems and better account for fish movement in complex landscapes with differential exposure pathways and conditions.