Abstract: This study attempted to molecular characterization of marine and coastal fishes of Bangladesh based on mitochondrial cytochrome c oxidase subunit I (COI) gene as a marker. A total of 376 mitochondrial COI barcode sequences were obtained from 185 species belonging to 146 genera, 74 families, 20 orders and two classes of fishes. The mean length of the sequences was 652 base pairs. For all the samples, %G was significantly lower compared to the other nucleotides and %GC was significantly lower compared to %AT (p ˂ 0.005). Also, a significantly lowered %GC content was observed in second and third codon position compared to the first codon position in all the samples (1st>2nd>3rd, p-value ˂ 0.005). In Elasmobranchii (Sharks and rays) the average Kimura two parameter (K2P) distances within species, genera, families and orders were 1.2%, 6.07%, 11.08% and 14.68%, respectively and for Actinopterygii, the average K2P distances within species, genera, families and orders were 0.40%, 6.36%, 14.10% and 24.07%, respectively. The mean interspecies distance was 16-fold higher than the mean intraspecies distance. The K2P neighbor-joining (NJ) trees based on the sequences generally clustered species in accordance with their taxonomic position. A total of 21 species were newly recorded in Bangladesh. High efficiency and fidelity in species identification and discrimination were demonstrated in the present study by DNA barcoding, and we concluded that COI sequencing can be used as an authentic identification marker for Bangladesh marine fish species. Key words: COI, Barcoding, Elasmobranchii, Actinopterygii, Genetic diversity, Phylogeny