The genetic barcode database constructed is made up of species of ray-finned (Actinopterygii) and cartilaginous (Elasmobranchii) fish defined on the basis of the bi-annual counts carried out as part of the National Coral Reef Monitoring Service by CRIOBE. In particular, the database contains information on the unique genetic signature or barcode of a small region of the mitochondrial 12S gene of each fish species from this inventory. The Actinopterygii-specific and Elasmobranchii-specific MiFish primers are two sets of primers that target a fragment of the mitochondrial 12S rRNA gene that is highly variable from one fish species to another. DNA extraction, PCR, and SANGER sequencing of the region of interest were performed on a collection of fish samples assembled and stored at CRIOBE. The resulting sequences were assembled with phrap (de la Bastide, M. and McCombie, W.R. (2007), Assembling Genomic DNA Sequences with PHRAP. Current Protocols in Bioinformatics, 17: 11.4.1-11.4.15. https://doi.org/10.1002/0471250953.bi1104s17) according to phred quality scores. To complement the laboratory approach , an in silico approach involved searching public databases for the 12S mitochondrial gene sequences of all fish. As the complete 12S sequence was not obtained for all species with our laboratory approach, and for the sake of uniformity, only the MiFish part of 12S was integrated into the database. The dataset includes sequences from 431 species belonging to the Actinopteri class and 15 species from the Elasmobranchi class. It also contains one Homo sapiens sequence, which is included to help identify and exclude potential contamination during taxonomic assignment analyses. The accompanying list_lineage file provides the complete taxonomy from NCBI (Species, Genus, Family, Order, Class, Phylum, Kingdom, Domain), with the NCBI accession number as the first column.