The U.S. Environmental Protection Agency and Environment Canada are supporting the development of indicators of ecosystem health that can be used to report on progress in restoring and maintaining the Great Lakes ecosystem, as called for in the Great Lakes Water Quality Agreement between the United States and Canada. One indicator under development is based on burrowing mayflies (Hexagenia: Ephemeroptera: Ephemeridae). We sampled in western Lake Erie, Saginaw Bay (Lake Huron), and Green Bay (Lake Michigan) in spring 2001 at 117 stations covering about 1,870 km² of lake bed, to determine the status of nymphal populations of Hexagenia, and to provide information that would further the technical development of an indicator of ecosystem health based on Hexagenia. In western Lake Erie, density and biomass of nymphs were generally highest on fine-grained substrate in offshore waters and were lower on coarser substrates in near shore waters. Nymphs were virtually absent from Saginaw Bay, where only one nymph was collected at 28 stations. Nymphs were collected at only 6 of 48 stations in Green Bay, and density and biomass were highest at the northern end of the bay. Polluted sediments are likely responsible for the absence or low density and biomass of nymphs observed on fine-grained substrates in western Lake Erie, Saginaw Bay, and Green Bay, all of which historically supported abundant populations.