The Barcoding was proposed motivated by the mismatch between the low number of taxonomists that contrasts with the large number of species, the method requires the construction of reference collections of DNA sequences that represent existing biodiversity. Freshwater fishes are key indicators for understanding biogeography around the world. Colombia with 1610 species of freshwater fishes is the second richest country in the world in this group. However, genetic information of the species continues to be limited, the contribution to a reference library of DNA barcodes for Colombian freshwater fishes highlights the importance of biological collections and seeks to strengthen inventories and taxonomy of such collections in future studies.

This dataset contributes to the knowledge on the DNA barcodes and occurrence records of 96 species of Freshwater fishes from Colombia. The species represented in this dataset correspond to an addition to BOLD public databases of 39 species. Forty-nine specimens were collected in Atrato bassin and 708 in Magdalena-Cauca bassin during the period of 2010 to 2020, two species (Loricariichthys brunneus and Poecilia sphenops) are considered exotic to the Atrato, Cauca and Magdalena basins and four species (Oncorhynchu mykiss, Oreochromis niloticus, Parachromis friedrichsthalii and Xiphophorus helleri) are exotic to Colombian hydrogeographic regions. All specimens are deposited in the CIUA collection at University of Antioquia and have their DNA barcodes made publicly available in the Barcode of Life Data System (BOLD) online database and the distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).