This U.S. Geological Survey (USGS) data release contains stream water chemistry and streamflow data collected in late August and early September, 2021 from 28 sites located throughout Colorado, USA. The sampled streams all drain high-elevation mountain watersheds in areas where the bedrock is hydrothermally altered and contains abundant sulfide minerals. Most sampled streams are therefore affected by natural acid-rock drainage. All sites had been sampled in prior years so that the 2021 data could be used to evaluate potential changes in stream water chemistry in recent decades. Streamflow was also quantified at most sites using data from a sodium chloride slug addition wherein specific conductivity readings were used as a surrogate for the tracer concentration.