Indonesia, home to a significant portion of the world’s fish biodiversity, faces challenges in accurately identifying and conserving its freshwater fish species, including Ompok hypophthalmus. This study examines the morphological and molecular identification status of O. hypophthalmus, highlighting inconsistencies in taxonomic practices and the need for integrative approaches. Morphological studies, such as those by Ng (2003), identified three distinct species within the O. hypophthalmus group, yet subsequent research often misclassified O. rhadinurus and O. urbaini as O. hypophthalmus. Molecular analyses, including DNA barcoding of cytochrome oxidase I (COI) and cytochrome b (Cyt b) genes, revealed significant genetic divergence among populations from different Indonesian rivers, suggesting speciation or misidentification in prior studies. Phylogenetic and genetic analyses confirmed the presence of O. hypophthalmus in Arut River, while Indragiri and Tapung Rivers were dominated by O. rhadinurus. These findings emphasize the importance of integrating morphological and genetic tools to resolve taxonomic ambiguities and support effective fish management and conservation strategies. This study provides critical genetic data, advocates for molecular methods in biodiversity assessments, and underscores the ecological significance of accurately identifying native and invasive fish species in Indonesia and World.

DNA barcoding is a technique in which species identification is performed by generating DNA barcodes from a small fragment of the mitochondrial genome. Here, molecular methods were used for assessment of 226 barcodes belonging to 83 fish species from 83 genera, 39 families and 21 orders of fishes with the average divergence within a species is 0.10%, 13.57% within a genus, and 17.33% within a family with 97–100% identity with comparison to the Genbank database and BOLD of the Chilika lagoon India, using, Barcode gap analysis, barcode index number and automatic barcode gap discovery, these methods had their potential to discriminate 97.53%, 93.90% and 95.06%, of species respectively. This is the first effort to generate the DNA barcode reference library of freshwater fishes from Chilika lagoon of India, one of the world’s biodiversity hotspots. These findings will contribute to the barcode database of global marine fish species identification, provide baseline data for fishes in Chilika Lagoon waters based on the CO1 region, and serve educational purposes in universities, research institutes, fishery managers, and fish stock assessment. The Barcode Index Number (BIN) discordance report showed that 226 specimens represented 83 BINs, of which 73.49%were taxonomically concordant and 26.50% were singletons and no discordant BIN found in our dataset.