Under the Great Lakes Restoration Initiative, the U.S. Geological Survey Mercury Research Lab (USGS MRL) conducted a multiyear assessment of mercury across the Laurentian Great Lakes. Biannual sampling was conducted across all five lakes onboard the U.S. Environmental Protection Agency (US EPA) research vessel Lake Guardian (in years 2010–2014, 2018) at pelagic sampling locations established by the long-term US EPA Great Lakes monitoring program. In addition to the regularly scheduled biannual sampling, in September 2013 and 2014 Lake Michigan and Lake Erie (respectively) were sampled with increased focus on shallow nearshore locations. Throughout these sampling efforts, sediments, mussels, surface water, and plankton were collected from open-water locations within all five of the Great Lakes. Additionally, surface water from Great Lake tributaries were sampled by the USGS MRL and preceded (2005 or 2006) or overlapped (2010-2015) with the open lake sampling efforts. All surface water samples were analyzed for methylmercury, total mercury, and dissolved organic carbon. Plankton and mussel tissue were analyzed for methylmercury, total mercury, and carbon/nitrogen stable isotopes and concentrations (plankton only). Sediments were analyzed for methylmercury, total mercury, and carbon content.