 We developed a set of universal PCR primers, MiFishUE, for metabarcoding environmental DNA, EDNA, from fishes. Primers were designed using aligned whole mitochondrial genome, mitogenome, sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmabranchs, sharks and rays. The primers target a hypervariable region of the 12S RNA gene, 163-185 BP, which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled EDNA from four tanks in the Okinawa Churomi Aquarium with known species compositions, prepared dual indexed libraries and performed paired end sequencing of the region using high throughput next generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species, 93.3% distributed across. This article was authored by M. Mia, Y. Soto, T. Fuquanaga, and others. We are article.tv, links in the description below.