: Zebra loach is one of the popular and desirable species in the ornamental sector that contributes to a major share for the world market of indigenous ornamental fish. Being categorized as Endangered under IUCN RedList, its identification has proved to be tricky owing to close variations in morphology and colour. The present study describes the application of morpho-taxonomy and genetic marker Cytochrome c Oxidase subunit I (COI) to understand the variations in Botia striata . COI gene (655 bp) was amplified using the universal primers and sequenced. A total of 10 samples were measured for standard length and the value ranged from 47.40 to 48.20 mm. Meristic characters i.e. , fin rays of dorsal, pectoral, pelvic, anal and caudal fins; and lateral transverse scales on the body were measured. For all the samples, the percentage of Thymine was significantly higher compared to the other nucleotides i.e. , 31.5%. The mean interspecific genetic distance among the species under study was found to be 0.037. The K2P based neighbor-joining (NJ) tree generated clustered the species according to their taxonomic position and showed that B. striata formed a separate clade (supported by 100% bootstrap value). The present study will be helpful in the identification of the Endangered and Vulnerable Botia species of Karnataka that will be beneficial for population studies, management and conservation programs. The present study also aids in developing a conservation-scheme approach for managing the fish diversity in their natural habitats.