The Little Blue River in Jackson County, Missouri, was listed as impaired in 2012 due to Escherichia coli (E. coli) from urban runoff and storm sewers. A study was initiated to characterize E. coli concentrations and loads to aid in the development of a total maximum daily load implementation plan. Longitudinal sampling along the stream revealed spatial and temporal variability in E. coli loads. Regression models were developed to better represent E. coli variability in the impaired reach using continuous hydrologic and water-quality parameters as predictive parameters. Daily loads calculated from main-stem samples were significantly higher downstream compared to upstream even though there was no significant difference between the upstream and downstream measured concentrations and no significant conclusions could be drawn from model-estimated loads due to model-associated uncertainty. Increasing sample frequency could decrease the bias and increase the accuracy of the modeled results.