Mercury (Hg) contamination of fisheries is a major concern for resource managers of many temperate lakes. Anthropogenic Hg contamination is largely derived from atmospheric deposition within a lake’s watershed, but its incorporation into the food web is facilitated by bacterial activity in sediments. Temporal variation in Hg content of fish (young-of-year yellow perch) in the regulated lakes of the Rainy–Namakan complex (on the border of the United States and Canada) has been linked to water level (WL) fluctuations, presumably through variation in sediment inundation. As a result, Hg contamination of fish has been linked to international regulations of WL fluctuation. Here we assess the relationship between WL fluctuations and fish Hg content using a 10-year dataset covering six lakes. Within-year WL rise did not appear in strongly supported models of fish Hg, but year-to-year variation in maximum water levels (∆maxWL) was positively associated with fish Hg content. This WL effect varied in magnitude among lakes: In Crane Lake, a 1 m increase in ∆maxWL from the previous year was associated with a 108 ng increase in fish Hg content (per gram wet weight), while the same WL change in Kabetogama was associated with only a 5 ng increase in fish Hg content. In half the lakes sampled here, effect sizes could not be distinguished from zero. Given the persistent and wide-ranging extent of Hg contamination and the large number of regulated waterways, future research is needed to identify the conditions in which WL fluctuations influence fish Hg content.