Despite historical observations of potential microcystin-producing cyanobacteria (including Leptolyngbya, Phormidium, Pseudoanabaena, and Anabaena species) in 74% of headwater streams in Alabama, Georgia, South Carolina, and North Carolina during 1993-2011, fluvial cyanotoxin occurrence has not been systematically assessed in the southeastern United States (USA). To begin to address this data gap, a spatial reconnaissance of fluvial microcystins (MC) concentrations was conducted in 75 wadeable streams in the Piedmont region during June 2014. Microcystins were detected using ELISA (limit = 0.10 µg/L) in 39% of the streams with mean, median, and maximum detected concentrations of 0.29, 0.11, and 3.2 µg/L, respectively. Significant (α = 0.05) correlations were observed between June 2014 MC concentrations and stream flow, total nitrogen to total phosphorus ratio (TN:TP), and water temperature, but each explained 38% or less of the variability in fluvial MC across the region. Temporal MC variability was assessed monthly through October 2014 in five of the streams where MC was observed in June and in one reference location; MC was repeatedly detected in all but the reference stream. While MC concentrations in this study did not exceed World Health Organization recreational guidance thresholds, their widespread occurrence demonstrates the need for further investigation of possible in-stream environmental health effects as well as potential impacts on downstream lakes and reservoirs. This article is protected by copyright. All rights reserved.