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Environmental DNA ( eDNA ) metabarcoding has been increasingly applied to biodiversity surveys in stream ecosystems. In stream networks, the accuracy of  eDNA -based biodiversity assessment depends on whether the upstream  eDNA  influx affects downstream detection. Biodiversity assessment in low-discharge streams should be less influenced by  eDNA  transport than in high-discharge streams. We estimated α- and β-diversity of the fish community from  eDNA  samples collected in a small Michigan (USA) stream from its headwaters to its confluence with a larger river. We found that α-diversity increased from upstream to downstream and, as predicted, we found a significant positive correlation between β-diversity and physical distance (stream length) between locations indicating species turnover along the longitudinal stream gradient. Sample replicates and different genetic markers showed similar species composition, supporting the consistency of the  eDNA  metabarcoding approach to estimate α- and β-diversity of fishes in low-discharge streams.

Estimating fish alpha- and beta-diversity along a small stream with environmental DNA metabarcoding