Identifying the sources of methylmercury (MeHg) and tracing the transformations of mercury (Hg) in the aquatic food web are important components of effective strategies for managing current and legacy Hg sources. In our previous work, we measured stable isotopes of Hg (δ²⁰²Hg, Δ¹⁹⁹Hg, and Δ²⁰⁰Hg) in the Laurentian Great Lakes and estimated source contributions of Hg to bottom sediment. Here, we identify isotopically distinct Hg signatures for Great Lakes trout (Salvelinus namaycush) and walleye (Sander vitreus), driven by both food-web and water-quality characteristics. Fish contain high values for odd-isotope mass independent fractionation (MIF) with averages ranging from 2.50 (western Lake Erie) to 6.18‰ (Lake Superior) in Δ¹⁹⁹Hg. The large range in odd-MIF reflects variability in the depth of the euphotic zone, where Hg is most likely incorporated into the food web. Even-isotope MIF (Δ²⁰⁰Hg), a potential tracer for Hg from precipitation, appears both disconnected from lake sedimentary sources and comparable in fish among the five lakes. We suggest that similar to the open ocean, water-column methylation also occurs in the Great Lakes, possibly transforming recently deposited atmospheric Hg deposition. We conclude that the degree of photochemical processing of Hg is controlled by phytoplankton uptake rather than by dissolved organic carbon quantity among lakes.