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The biodiverse Neotropical ecoregion remains insufficiently assessed, poorly managed, and threatened by unregulated human activities. Novel, rapid and cost-effective DNA-based approaches are valuable to improve understanding of the biological communities and for biomonitoring in remote areas. Here, we evaluate the potential of environmental DNA (eDNA) metabarcoding for assessing the structure and distribution of fish communities by analysing water and sediment from 11 locations along the Jequitinhonha River catchment (Brazil). Each site was sampled twice, before and after a major rain event in a five-week period and fish diversity was estimated using high-throughput sequencing of 12S rRNA amplicons. In total, 252 Molecular Operational Taxonomic Units (MOTUs) and 34 fish species were recovered, including endemic, introduced, and previously unrecorded species for this basin. Spatio-temporal …