Great Lakes tributaries are known to deliver waterborne pathogens from a host of sources. To examine the hydrologic, land cover, and seasonal patterns of waterborne pathogens (i.e. protozoa (2), pathogenic bacteria (4) human viruses, (8) and bovine viruses (8)) eight rivers were monitored in the Great Lakes Basin over 29 months from February 2011 to June 2013. Sampling locations represented a wide variety of land cover classes from urban to agriculture to forest. A custom automated pathogen sampler was deployed at eight sampling locations which provided unattended, flow-weighted, large-volume (120-1630 L) sampling. Human and bovine viruses and pathogenic bacteria were detected by real-time qPCR in 16%, 14%, and 1.4% of 290 samples collected while protozoa were never detected. The most frequently detected pathogens were: bovine polyomavirus (11%), and human adenovirus C, D, F (9%). Human and bovine viruses were present in 16.9% and 14.8% of runoff-event samples (n = 189) resulting from precipitation and snowmelt, and 13.9% and 12.9% of low-flow samples (n = 101), respectively, indicating multiple delivery mechanisms could be influential. Data indicated human and bovine virus prevalence was different depending on land cover within the watershed. Occurrence, concentration, and flux of human viruses were greatest in samples from the three sampling locations with greater than 25% urban influence than those with less than 25% urban influence. Similarly, occurrence, concentration, and flux of bovine viruses were greatest in samples from the two sampling locations with greater than 50 cattle/km(2) than those with less than 50 cattle/km(2). In seasonal analysis, human and bovine viruses occurred more frequently in spring and winter seasons than during the fall and summer. Concentration, occurrence, and flux in the context of hydrologic condition, seasonality, and land use must be considered for each watershed individually to develop effective watershed management strategies for pathogen reduction.