In 2013, the first of several Regional Stream Quality Assessments (RSQA) was done in the Midwest United States. The Midwest Stream Quality Assessment (MSQA) was a collaborative study by the U.S. Geological Survey (USGS) National Water Quality Assessment (NAWQA), the USGS Columbia Environmental Research Center, and the U.S. Environmental Protection Agency (USEPA) National Rivers and Streams Assessment (NRSA). One of the objectives of the RSQA, and thus the MSQA, is to characterize the relationships between water-quality stressors and stream ecology and to determine the relative effects of these stressors on aquatic biota within the streams (U.S. Geological Survey, 2012a). To meet this objective, a framework of fundamental geospatial data was required to develop physical and anthropogenic characteristics of the study region, sampled sites and corresponding watersheds, and riparian zones. This dataset represents the boundaries for the 100 watersheds studied in the MSQA, and is one of the four fundamental geospatial data layers that were developed for the Midwest study.