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Fecal contamination from sewage and agricultural runoff is a pervasive problem in Great Lakes watersheds. Most work examining fecal pollution loads relies on discrete samples of fecal indicators and modeling land use. In this study, we made empirical measurements of human and ruminant-associated fecal indicator bacteria and combined these with hydrological measurements in eight watersheds ranging from predominantly forested to highly urbanized. Flow composited river samples were collected over low-flow (n = 89) and rainfall or snowmelt runoff events (n = 130). Approximately 90% of samples had evidence of human fecal pollution, with highest loads from urban watersheds. Ruminant indicators were found in ∼60–100% of runoff-event samples in agricultural watersheds, with concentrations and loads related to cattle density. Rain depth, season, agricultural tile drainage, and human or cattle density explained variability in daily flux of human or ruminant indicators. Mapping host-associated indicator loads to watershed discharge points sheds light on the type, level, and possible health risk from fecal pollution entering the Great Lakes and can inform total maximum daily load implementation and other management practices to target specific fecal pollution sources.