Natural flow regimes represent the hydrologic conditions to which native aquatic organisms are best adapted. We completed a regional river classification and quantitative descriptions of each natural flow regime for the Ozark–Ouachita Interior Highlands region of Arkansas, Missouri and Oklahoma. On the basis of daily flow records from 64 reference streams, seven natural flow regimes were identified with mixture model cluster analysis: Groundwater Stable, Groundwater, Groundwater Flashy, Perennial Runoff, Runoff Flashy, Intermittent Runoff and Intermittent Flashy. Sets of flow metrics were selected that best quantified nine ecologically important components of these natural flow regimes. An uncertainty analysis was performed to avoid selecting metrics strongly affected by measurement uncertainty that can result from short periods of record. Measurement uncertainties (bias, precision and accuracy) were assessed for 170 commonly used flow metrics. The ranges of variability expected for select flow metrics under natural conditions were quantified for each flow regime to provide a reference for future assessments of hydrologic alteration. A random forest model was used to predict the natural flow regimes of all stream segments in the study area based on climate and catchment characteristics, and a map was produced. The geographic distribution of flow regimes suggested distinct ecohydrological regions that may be useful for conservation planning. This project provides a hydrologic foundation for future examination of flow–ecology relationships in the region. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.