Background. on a short standardized segment of the genome. The main objective of the barcode of life project is to provide a database of genetic sequences which can be used as a tool for universal species identification. Indonesia has at least 1300 freshwater fish species, however unfortunately no species has been barcoded as yet. In the presently reported study, we subjected to barcoding a total of 14 species of freshwater fishes from Lake Laut Tawar, Indonesia. Materials and Methods. On average, 10 random samples from each species were processed for DNA analysis. Approximately 655-bp were amplified from the 5′ region of the mitochondrial cytochrome C oxidase subunit I (COX1) gene. All obtained sequences were edited and aligned using MEGA 4.0 program. Nucleotide divergence among sequences was estimated based on Kimura 2-parameter distances. Unique haplotypes were determined using DnaSP Version 5.10.02 software, and the genetic relations among haplotypes were assessed by constructing a phenogram using the neighbour-joining method. Results. A total of 31 haplotypes from 14 freshwater fish species were produced in this study. The read lengths were 626-bp, where 259 sites were polymorphic, 254 sites parsimony informative, and five singletons. No stop codons, deletions, or insertions were observed in any of the sequences. The nucleotide distance between species ranged from 7.1%—between Puntius brevis (Bleeker, 1850) and Poropuntius tawarensis (Weber et de Beaufort, 1916)—to 30.4%—between Channa gachua (Hamilton, 1822) and Homaloptera sp.—indicating that P. brevis and P. tawarensis are very closely related. Conclusion. >This study confirms the utility of COX1 gene in accurate identification of 14 species of freshwater fishes from Lake Laut Tawar, however, three species could not be identified to species level namely Rasbora sp. (local name: relo), Homaloptera sp. (ilie) and Clarias sp. (mud). It is suggested that future studies should incorporate morphometric methods to resolve the taxonomic status of these undetermined species
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