Hydrologic, seasonal, and spatial variability of sewage contamination was studied at six locations within a watershed upstream from water reclamation facility (WRF) effluent to define relative loadings of sewage from different portions of the watershed. Fecal pollution from human sources was spatially quantified by measuring two human-associated indicator bacteria (HIB) and eight human-specific viruses (HSV) at six stream locations in the Menomonee River watershed in Milwaukee, Wisconsin from April 2009 to March 2011. A custom, automated water sampler, which included HSV filtration, was deployed at each location providing unattended, flow-weighted, large-volume (30-913 L) sampling. In addition, wastewater influent samples were composited over discrete seven-day periods from the two Milwaukee WRFs. Of the eight HSV only three were detected, present in up to 38% of the 228 stream samples, while at least one HSV was detected in all WRF influent samples. HIB occurred more often with significantly higher concentrations than the HSV in stream and WRF influent samples (p<0.05). HSV yield calculations showed a loss from upstream to the most downstream sub-watershed of the Menomonee River, and in contrast, a positive HIB yield from this same sub-watershed emphasizes the complexity in fate and transport properties between HSV and HIB. This study demonstrates the utility of analyzing multiple HSV and HIB to provide a weight of evidence approach for assessment of fecal contamination at the watershed level, provides an assessment of relative loadings for prioritizing areas within a watershed, and demonstrates how loadings of HSV and HIB can be inconsistent, inferring potential differences in fate and transport between the two indicators of human fecal presence.