Urea can degrade water quality and stimulate toxic phytoplankton in P-rich lakes, yet little is known of its sources, abundance, or transportation in lotic systems, particularly within the Northern Great Plains. We measured physico-chemical parameters biweekly during May–September 2010–2012 at 16 stations along a 250 km lotic continuum to quantify spatial and temporal variation in urea concentrations and discharge, and to identify potential regulatory processes. Urea concentrations were similar to those in regional prairie lakes (range 5.2–792.1, median 78.6 μg N L−1) with variable seasonal mean (± SD) concentrations (96.6  ± 96.1 μg N L−1) and fluxes (4.22 × 105 ± 257.6 μg N s−1). Landscape analysis with generalized additive models explained 68.3% of deviance in urea concentrations, with high temporal variability predicted mainly by positive relationships with nutrient content and chlorophyte abundance, but not temperature, dissolved organic matter, bacterial abundance, or urban effluent. Seasonal analysis revealed that during spring, urea content was correlated negatively with leguminous forage cover (% area) and positively with stream discharge, oilseed and cereal crops, and shrubs or deciduous plants, while during summer, urea concentrations were correlated negatively with discharge and leguminous crop cover, as well as nutrient levels. Mean porewater urea concentrations (528.5 ± 229.8 μg N L−1) were over five-fold greater than stream concentrations, suggesting that hyporheic production may offset declining influx from terrestrial sources during summer. We conclude that urea may be ubiquitous in eutrophic prairie streams and that management of its export from land may reduce detrimental effects on downstream lakes.