The study sequenced the mitochondrial cytochrome oxidase subunit I (COI) gene for 207 fish species, primarily Australian marine fish, to evaluate the effectiveness of DNA barcoding for species identification. A total of 754 sequences were generated from multiple specimens of most species. The average within-species, genus, family, order, and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18%, and 23.27%, respectively. All species could be differentiated by their COI sequences, although some congeneric individuals showed deep divergences. The study found that COI sequencing can effectively identify fish species, with higher GC content observed in teleosts compared to sharks and rays. The results also revealed phylogenetic signals, with four major clusters in the neighbor-joining tree: chimaeras, rays, sharks, and teleosts. Detailed analyses of flatheads, tunas, and dogfishes showed high species-level differentiation, supporting the utility of COI barcoding for species identification in these groups. The study concludes that COI sequencing is a reliable method for identifying fish species, with potential applications in fisheries management, ecosystem research, and conservation.The study sequenced the mitochondrial cytochrome oxidase subunit I (COI) gene for 207 fish species, primarily Australian marine fish, to evaluate the effectiveness of DNA barcoding for species identification. A total of 754 sequences were generated from multiple specimens of most species. The average within-species, genus, family, order, and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18%, and 23.27%, respectively. All species could be differentiated by their COI sequences, although some congeneric individuals showed deep divergences. The study found that COI sequencing can effectively identify fish species, with higher GC content observed in teleosts compared to sharks and rays. The results also revealed phylogenetic signals, with four major clusters in the neighbor-joining tree: chimaeras, rays, sharks, and teleosts. Detailed analyses of flatheads, tunas, and dogfishes showed high species-level differentiation, supporting the utility of COI barcoding for species identification in these groups. The study concludes that COI sequencing is a reliable method for identifying fish species, with potential applications in fisheries management, ecosystem research, and conservation.