This study reports the application of Soil and Water Assessment Tool (SWAT) within the Hydrologic and Water Quality System (HAWQS) on-line platform, for the Upper Mississippi River Basin (UMRB). The UMRB is an important ecosystem located in the north-central U.S. that is experiencing a range of ecological stresses. Specifically, testing of SWAT was performed for: (1) Hargreaves (HG) and Penman-Monteith (PM) PET methods, and (2) Livneh, National Climatic Data Center (NCDC) and Parameter-elevation Regressions on Independent Slopes Model (PRISM) climate datasets. The Livneh-PM combination resulted in the highest average annual water yield of 380.6 mm versus the lowest estimated water yield of 193.9 mm for the Livneh-HG combination, in response to 23-year uncalibrated simulations. Higher annual ET and PET values were predicted with HG method versus the PM method for all three weather datasets in response to the uncalibrated simulations, due primarily to higher HG-based estimates during the growing season. Based on these results, it was found that the HG method is the preferred PET option for the UMRB. Initial calibration of SWAT was performed using the Livneh data and HG method for three Mississippi River main stem gauge sites, which was followed by spatial validation at 10 other gauge sites located within the UMRB stream network. Overall satisfactory results were found for the calibration and validation gauge sites, with the majority of R2 values ranging between 0.61 and 0.82, Nash-Sutcliffe modeling efficiency (NSE) values ranging between 0.50 and 0.79, and Kling-Gupta efficiency (KGE) values ranging between 0.61 and 0.84. The results of an additional experimental suite of six scenarios, which represented different combinations of climate data sets and calibrated parameters, revealed that suggested statistical criteria were again satisfied by the different scenario combinations. Overall, the PRISM data exhibited the strongest reliability for the UMRB.