The current study represents the first molecular characterization of freshwater fish species in Egypt from two major fish resources; the River Nile and Lake Nasser. A total of 160 DNA barcodes using a 655-bp-long fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene were generated from 37 species belonging to 32 genera that represent 15 families from nine orders. The studied species were identified using different molecular-based identification approaches, in addition to the morphological identification, including neighbor-joining (NJ) trees, Barcode Index Number, and Automatic Barcode Gap Discovery (ABGD). The average genetic divergence based on the Kimura two-parameter model (K2P) within orders, families, genera, and species were 0.175, 0.067, 0.02, and 0.008, respectively. The minimum and maximum K2P distance-based genetic divergences were 0.0 and 0.154, respectively. Nucleotide diversity (π) varied among families and ranged between 0.0% for families Malapteruridae, Auchenoglanididae, Schilbeidae, Anguillidae, Centropomidae and Tetraodontidae and 17% for family Cyprinidae. The current study cautions against the lack of species coverage at public databases which limits the accurate identification of newly surveyed species and recommends that multiple methods are encouraged for accurate species identification. The findings of the current study also support that COI barcode enabled effective fish species identification in River Nile and Lake Nasser. Moreover, the results of the current study will establish a comprehensive DNA barcode library for freshwater fishes along the River Nile in Egypt. Egyptian freshwater fish DNA barcodes will contribute substantially to future efforts in monitoring, conservation, and management of fisheries in Egypt.