NADH dehydrogenase is a very important protein and is expressed by the mitochondrial NADH dehydrogenase gene (mtND2). Dehydrogenase enzyme is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP. Also, it reversibly catalyses the oxidation of NADH to NAD and reduced acceptor. The size of mtDN2 of Tilapia species and their hybrids is ~1050 base pairs and was detected by using the polymerase chain reaction technique. To identify the molecular phylogeny and the physical characteristics of mtND2 gene of Tilapia species were done by using the restriction fragment length polymorphisms (RFLPs) with some restriction endonucleases(AccI, AvaII, AvaI, StyI, Bg1I and EaeI). The PCR-RFLPs of NADH dehydrogenase gene of Tilapia species and their hybrids may prove that the gene is quite evolution phylogenetic difference from one species to another. At the same time, This study investigated the feasibility of mitochondrial DNA (mtDNA) based approaches in addressing problems of identification of Tilapia species and their hybrids, isolated from the River Nile by using the PCR-RFLPs analysis of mtND2 gene.