Technical Abstract: Fecal bacteria contamination of surface waters continues to be a critical water quality concern with serious human health implications, but relatively few land use specific data sets are available to guide management, restoration, policy, and regulatory decisions. In regions with substantial poultry production, litter application sites are often assumed to be major contributors to bacterial contamination, and grazing lands often receive a similar focus. Since most states use Escherichia coli (E. coli) as an indicator organism for fecal contamination, this study was designed to measure E. coli concentrations in runoff from small agricultural watersheds with various land uses. Specifically, 3 yr of water quality data were collected from 13 watersheds and analyzed to evaluate the impacts of litter application and land use on runoff E. coli concentrations. In this study, litter application did not impact E. coli concentrations in runoff, which can at least partially be attributed to late summer application of litter that was produced and removed from poultry houses during hot, dry conditions unfavorable for E. coli survival. In terms of land use impacts, cultivated fields with and without litter application produced the lowest E. coli concentrations in runoff presumably due to limited wildlife presence. In contrast, the native prairie reference site produced relatively high E. coli concentrations due to increased fecal deposition presumably from abundant wildlife. The high concentrations of E. coli from grazed lands emphasize the need for livestock producers to follow best management practice recommendations to minimize their bacteria contribution; however, it is important to note that high E. coli concentrations were measured in runoff from well-managed grazing lands as well as ungrazed native prairie, which indicates the difficulty of managing bacterial contamination.