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 National Water Quality Assessment and the U.S. Environmental Protection Agency National Rivers and Streams Assessment. One of the objectives of the RSQA, and thus the MSQA, is to characterize relations between stream ecology and water-quality stressors to determine the relative effects of these stressors on aquatic biota in streams. Data required to meet this objective included fish species and abundance data and physical and chemical water-quality characteristics of the ecological reaches of the sites that were sampled. This dataset comprises 135 fish species, 39,920 fish, 10 selected water-quality stressor metrics, and six selected fish community stressor response variables for 98 sites sampled for the MSQA.