Fish is an extremely nutritious food that is abundant in important minerals, vitamin D, protein, and omega-3 fatty acids. Now a day, mislabeling of fish products is a major problem in the fish industry that affects the market prices and causes fluctuations. Phylogenetic analysis of nucleotide sequences is a technique that might be used to verify the identity of misidentified fish species. In this study five species (Cirrhinus mrigala, Hypophthalmichthys molitrix, two Actinopterygii sp., and Mastacembelus armatus) were used for phylogenetic analysis using the COI gene was amplified using the PCR, average product size is 658 base pairs. A comparison of obtained sequences was implemented through National Center for Biotechnology Information database (NCBI) and the Basic Local Alignment Search Tool (BLAST). Most resemble sequences were downloaded for comparison with our sequencing results. By using MAGA X the rates of different transitional substitutions are 11.33 and transversional substitutions are 6.83. The estimated Transition/Transversion bias (R) is 0.83. The K2P model was used for the estimation of this substitution pattern. Because most freshwater fish species were in the same order or family, phylogenetic tree analysis indicated that they grouped together. Using the InterPro database, the activity and regulatory pathways of the Cytochrome C Oxidase 1 gene are identified to show their activity on the protein level, especially for some important physiological functions. Each species of fish could be identified based on their speciation and node position and distance from their parent nodes. Based on the closest phylogenetic relations, DNA sequence-based study was developed for the accurate identification of fish species.