DNA barcoding was used to identify 207 fish species, mostly Australian marine fish, by sequencing a 655 bp region of the mitochondrial cytochrome oxidase subunit I (cox1) gene. A total of 754 sequences were generated, with 143 teleost species showing higher GC content (47.1%) than sharks and rays (42.2%), largely due to higher GC in the third codon position. Species could be differentiated by cox1 sequences, though some had haplotypes similar to congeneric species. DNA barcoding proved effective in identifying fish species, with clear phylogenetic signals in the data. The analysis revealed four major clusters: chimaerids, rays, sharks, and teleosts. Three taxonomic groups—dogfishes, flatheads, and tunas—were examined in detail, showing distinct genetic divergence. The study supports the use of cox1 sequencing for fish species identification, highlighting its potential for unambiguous identification of fish species, including eggs, larvae, fillets, and fins. The results indicate that DNA barcoding is a reliable method for species identification, with high genetic divergence among species and low within-species variation. The study also highlights the importance of accurate species identification in barcode library development and the need to retain voucher specimens for future analysis. The findings suggest that DNA barcoding can be a powerful tool for fish identification, with potential applications in fisheries management, conservation, and research.DNA barcoding was used to identify 207 fish species, mostly Australian marine fish, by sequencing a 655 bp region of the mitochondrial cytochrome oxidase subunit I (cox1) gene. A total of 754 sequences were generated, with 143 teleost species showing higher GC content (47.1%) than sharks and rays (42.2%), largely due to higher GC in the third codon position. Species could be differentiated by cox1 sequences, though some had haplotypes similar to congeneric species. DNA barcoding proved effective in identifying fish species, with clear phylogenetic signals in the data. The analysis revealed four major clusters: chimaerids, rays, sharks, and teleosts. Three taxonomic groups—dogfishes, flatheads, and tunas—were examined in detail, showing distinct genetic divergence. The study supports the use of cox1 sequencing for fish species identification, highlighting its potential for unambiguous identification of fish species, including eggs, larvae, fillets, and fins. The results indicate that DNA barcoding is a reliable method for species identification, with high genetic divergence among species and low within-species variation. The study also highlights the importance of accurate species identification in barcode library development and the need to retain voucher specimens for future analysis. The findings suggest that DNA barcoding can be a powerful tool for fish identification, with potential applications in fisheries management, conservation, and research.