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¹ Dr. Babasaheb Ambedkar Marathwada University, Paul Hebert Centre for DNA Barcoding & Biodiversity Studies, Aurangabad, Maharashtra, India, 431003
² Dr. Babasaheb Ambedkar Marathwada University, Department of Zoology, Aurangabad, Maharashtra, India, 431003 DOI : 10.4194/GA1021 Viewed : 30 - Downloaded : 35 Freshwater fish diversity in the Mahanadi River basin remains poorly resolved at the molecular level, and the absence of a curated DNA barcode reference library has limited accurate species identification, biodiversity monitoring, and conservation planning in this ecologically important system. To address this gap, we generated a comprehensive COI-based DNA barcode dataset comprising 421 specimens representing 121 morphologically identified species collected across eight locations in the basin. Species delimitation analyses revealed high concordance among taxa, with most species forming distinct genetic clusters and a clear barcode gap that is mean intraspecific divergence was 0.20% and interspecific divergence around 11.84% confirming the effectiveness of COI for species discrimination. Notably, multiple taxa exhibited deep intraspecific divergence, indicating the presence of potential cryptic diversity within the basin. In addition, several non-native species were detected, highlighting ongoing biotic homogenization and emerging ecological risks to native fish communities. This study establishes the first basin-scale DNA barcode reference library for the Mahanadi River and reveals previously underrecognized lineage-level diversity. The dataset provides a critical molecular baseline for future taxonomic revision, environmental DNA (eDNA)-based monitoring, and evidence-based conservation and fisheries management in tropical freshwater ecosystems. Keywords : DNA barcoding, Biodiversity, Mahanadi river, Species delimitation, Conservation