This dataset was collected to inform the Water, Sustainability and Climate Minnesota River Basin Observatory, and was supported by the National Science Foundation under Grant No. 1209402 Water, Sustainability and Climate (WSC) – Category 2, Collaborative: Climate and human dynamics as amplifiers of natural change: a framework for vulnerability assessment and mitigation planning. The dataset contains point locations, watershed areas and water quality information for 231 ditch, stream, river and wetland sites located in the Le Sueur River, Chippewa River, Cottonwood River, Cannon River, Wantonwan River and Blue Earth River basins of Minnesota. Study sites ranged in size from 1st order ditches and streams to an 8th order river. Each of these sites was sampled at least once between 2013-2016 (most sites were sampled multiple times) for one or more of the following parameters: 1) water chemistry (total dissolved nitrogen, nitrate-N, nitrite-N, ammonium-N, particulate nitrogen, soluble reactive phosphorus, total dissolved phosphorus, particulate phosphorus, total phosphorus, dissolved organic carbon, dissolved inorganic carbon, particulate carbon, chlorophyll a, total suspended solids, volatile suspended solids, delta-H-2 and delta-O-18 stable isotopes of site water, specific UV absorbance (SUVA) of site water, fluorescence index (FI) of site water); 2) stable isotopes (delta-C-13, delta-N-15, delta-H-2) of invertebrate consumers, particulate carbon and potential food sources; 3) denitrification rates and characteristics of benthic sediment in agricultural drainage ditches; and 4) stream discharge. This dataset also includes spatial data files containing study site locations and watershed areas delineated for each site.