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.