Using the Soil and Water Assessment Tool (SWAT) for large-scale watershed modeling could be useful for evaluating the quality of the water in regions that are dominated by nonpoint sources in order to identify potential "hot spots" for which mitigating strategies could be further developed. An analysis of water quality under future scenarios in which changes in land use would be made to accommodate increased biofuel production was developed for the Missouri River Basin (MoRB) based on a SWAT model application. The analysis covered major agricultural crops and biofuel feedstock in the MoRB, including pasture land, hay, corn, soybeans, wheat, and switchgrass. The analysis examined, at multiple temporal and spatial scales, how nitrate, organic nitrogen, and total nitrogen; phosphorus, organic phosphorus, inorganic phosphorus, and total phosphorus; suspended sediments; and water flow (water yield) would respond to the shifts in land use that would occur under proposed future scenarios. The analysis was conducted at three geospatial scales: (1) large tributary basin scale (two: Upper MoRB and Lower MoRB); (2) regional watershed scale (seven: Upper Missouri River, Middle Missouri River, Middle Lower Missouri River, Lower Missouri River, Yellowstone River, Platte River, and Kansas River); and (3) eight-digit hydrologic unit (HUC-8) subbasin scale (307 subbasins). Results showed that subbasin-level variations were substantial. Nitrogen loadings decreased across the entire Upper MoRB, and they increased in several subbasins in the Lower MoRB. Most nitrate reductions occurred in lateral flow. Also at the subbasin level, phosphorus in organic, sediment, and soluble forms was reduced by 35%, 45%, and 65%, respectively. Suspended sediments increased in 68% of the subbasins. The water yield decreased in 62% of the subbasins. In the Kansas River watershed, the water quality improved significantly with regard to every nitrogen and phosphorus compound. The improvement was clearly attributable to the conversion of a large amount of land to switchgrass. The Middle Lower Missouri River and Lower Missouri River were identified as hot regions. Further analysis identified four subbasins (10240002, 10230007, 10290402, and 10300200) as being the most vulnerable in terms of sediment, nitrogen, and phosphorus loadings. Overall, results suggest that increasing the amount of switchgrass acreage in the hot spots should be considered to mitigate the nutrient loads. The study provides an analytical method to support stakeholders in making informed decisions that balance biofuel production and water sustainability.