Background: The present investigation was carried out on the development of a barcode and DNA sequences database of Oreochromis niloticus (Nile Tilapia) of Shahpura Lake, Bhopal through mitochondrial Cytochrome c Oxidase-I gene (cox1) for public domain uses as a reference database for identification, authentication and variation studies. Materials and Methods: We performed the mitochondrial genomic analyses for molecular studies of which genomic DNA was extracted from fish tissues using the standard protocol provided by Janarthanan and Vincent (2007). Then, isolated DNA was introduced to Polymerase Chain Reaction (PCR) using universal primers, after which, the electrophoresis of the PCR product was done, then, obtained the DNA bands of interest of desired molecular weight on the gel. Results: We generated 02 unique DNA barcodes of morphologically identified fish specimens collected from Shahpura Lake, Bhopal. Considering the ambiguous (0%), Barcode Index Number (BIN) URI (BOLD:AAC9904), Top Hit % (100%), Nearest Member of Neighbor (ANGBF54446-19), Nearest BIN URI (BOLDAET5315), Analysis of Barcode Gap as an average intra-specific (204.14) and Analysis of Cluster Sequences (RESL) (20.583717) was found significant for development of DNA barcode of O. niloticus. The overall mean distance among O. niloticus specimens (6A and 6B) was obtained as 1.82 which may be considered as good for conservation point of view. RESL in the BOLD systems has a stronger taxonomic performance than that of the Barcode Gap Analysis and thus showed better species identification, during the present investigation achieved similar results, which may be related to the species identification.
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