Fasciolosis is an important food and water-borne parasitic infection caused by the two trematode species, Fasciola hepatica, and F. gigantica. The present study aimed to identify the phenotypic features and genetic characterization of adult fasciolid that infecting buffaloes were studied in Aswan, Egypt. The genetic identity of Fasciola species was investigated by the analysis of forward and reverse sequences of the ITS-2 of the rDNA gene. The Fasciola isolates were obtained from sheep, buffaloes, and cows in the regions of Aswan. The sequence of ITS2 gene isolates obtained from the present investigation were compared with GenBank reference sequences of F. hepatica, F. gigantica, and intermediate Fasciola. The obtained results were based on morphometric and genetic data which revealed the existence of F. gigantica, F. hepatica, and an intermediate form of Fasciola. Several variable sites were encountered among the investigated isolates in the Aswan, that were compared with the Fasciola species acquiesced in Gene Bank. Furthermore, the relationships between Egyptian Fasciola and Fasciola spp. from various other nations were discussed in the study.