The study of the Nile Puffer Fish (Tetraodon lineatus) from Lake Nasser, Aswan, Egypt is still at a very early stage. Samples from two locations (Khor El Ramla and Tushka East) from Lake Nasser were studied. Partial sequences of the COI gene (barcode) for 20 Nile Puffer Fish from Lake Nasser were successfully amplified and analyzed. The average of nucleotides composition percent of both locations samples was 25.88, 31.54, 24.53, and 18.05 for T, C, A, and G respectively. The base composition analysis of both locations samples of the COI sequence revealed that AT content (50.41%) was higher than GC (49.59%). The current results of the phylogenetic analysis of Lake Nasser samples indicate that there are three groups of Nile Pufferfish in Lake Nasser. Two of them are presented in the south and the last one in the north and south of the Lake. Conversely, the GenBank and Lake Nasser samples indicate that there are two haplotypes from Tetraodon lineatus of the two locations from Lake Nasser based on the partial sequence of the COI gene. The first haplotype includes different samples from the two studied locations (North and south Lake) and the second includes five samples from the North Lake. Finally, the present phylogenetic results for Nile Pufferfish in Lake Nasser (Tetraodon lineatus), together with other African freshwater groups, matched with the monophyletic origin of African freshwater Puffer fishes. As a conclusion, overall data collected from this study will be fundamental for the Lake Nasser fishery and the handling of the problems caused by Nile pufferfish. Therefore, proper utilization of this species towards sustainable management, nutritional composition, and biosecurity issues will lead to achieving a sustainable blue economy.