In 2013, the Regional Stream Quality Assessment (RSQA) study was started as part of the U.S. Geological Survey (USGS) National Water Quality Assessment (NAWQA) project. One of the objectives of the RSQA is to characterize the relationships between water-quality stressors and stream ecology and subsequently determine the relative effects of these stressors on aquatic biota within the streams (Garrett and others, 2017; Journey and others, 2015; Coles and others, 2019; Sheibley and others, 2017; May and others, 2020). The study was implemented in five regions across the United States (U.S.); the Midwest (MSQA) in 2013, the Southeast (SESQA) in 2014, the Pacific Northwest (PNSQA) in 2015, the Northeast (NESQA) in 2016, and California (CSQA) in 2017. To meet this objective, a framework of fundamental geospatial data was required to develop physical and anthropogenic characteristics of each study region, sampled sites, and corresponding watersheds and riparian zones. This dataset represents the location of the 483 water-quality sampling sites within the five regional study areas sampled for the RSQA and is one of the four fundamental geospatial data layers that were developed for the regional study.