Cyprinion watsoni is fresh water fish commonly known as Indus lotak or Watson cyprinid is native fish to the Indus River basin, Pakistan, but also found in Afghanistan, Iraq, Iran, Arabian Peninsula and Oman. This fish inhibits a verities of aquatic environments i.e. rivers, deep water pools, lakes, streams, and brackish water. A field survey of the present study was conducted in August, 2024 to investigate the morphological and genetic relationship of C. watsoni Population using Cytochrome oxidase gene sub unit I (COI) from Indus River, district Swabi, Khyber Pakhtunkhwa, Pakistan. A fish sample were collected from Tarbela region of the Indus River and morphologically identified as Cyprinion species using standard taxonomic key. We sequenced and submitted a Cytochrome oxidase sub unit I (COI) gene to GenBank with accession number PV444642 from first time of Pakistan. The Phylogenetic analysis of the reported COI gene from Pakistan showed high similarity of almost 91.34% with C. watsoni reported from Saudi Arabia. For further conformation, we establish a Median joining haplotype network and calculate the nucleotide and haplotype diversity of COI gene. The calculated Haplotypes diversity (hd) was 0.9848 and nucleotide diversity (π) was 0.0801865.