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Metabarcoding of bulk or environmental DNA has great potential for biomonitoring of freshwater environments. However, successful application of metabarcoding to biodiversity monitoring requires universal primers with high taxonomic coverage that amplify highly variable, short metabarcodes with high taxonomic resolution. Moreover, reliable and extensive reference databases are essential to match the outcome of metabarcoding analyses with available taxonomy and biomonitoring indices. Benthic invertebrates, particularly insects, are key taxa for freshwater bioassessment. Nevertheless, few studies have so far assessed markers for metabarcoding of freshwater macrobenthos. Here we combined in silico and laboratory analyses to test the performance of different markers amplifying regions in the 18S rDNA (Euka02), 16S rDNA (Inse01) and COI (BF1_BR2-COI) genes, and developed an extensive database of benthic macroinvertebrates of France and Europe, with a particular focus on key insect orders (Ephemeroptera, Plecoptera and Trichoptera). Analyses on 1,514 individuals representing different taxa of benthic macroinvertebrates showed very different amplification success across primer combinations. The Euka02 marker showed the highest universality, while the Inse01 marker showed excellent performance for the amplification of insects. BF1_BR2-COI showed the highest resolution, while the resolution of Euka02 was often limited. By combining our data with GenBank information, we developed a curated database including sequences representing 822 genera. The heterogeneous performance of the different primers highlights the complexity in identifying the best markers, and advocates for the integration of multiple metabarcodes for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity., (© 2020 John Wiley & Sons Ltd.)