Identification of liver fluke species cannot be achieved by clinical, pathological, coprological or immunological methods. However, the differential diagnosis between F. hepatica and F. gigantica infection is very important because of their different pathological manifestations. Moreover, in countries where the two species co-exist, morphologically intermediate forms were reported. The present study aimed to identify these forms by the use of molecular characterization of DNA sequence. Based on morphometric criteria, adults of Fasciola hepatica, F. gigantica and intermediate forms were collected from naturally infected sheep and cattle from various regions of Sohag Governorate. A simple and rapid new method (QIAamp DNA Mini Kit) was used to isolate DNA from the worms and their RELP patterns were obtained after digestion of the PCR products with AvaII restriction enzymes. The result of a regular PCR experiment for the amplification of the selected 28S rDNA fragment with the designed primer set yielded identical 618- bp-long PCR products for the three types of Fasciola where the RFLP profile obtained from F. hepatica revealed four fragments of 628, 575, 165 and 95 bp, and F. gigantica generated three fragments corresponding to 628, 358 and 300 bp fragments whereas the intermediate forms revealed four fragments of 628, 541, 358 and 300 bp, which were similar to those of F. gigantica but with a distinctive fragment of 541. These results confirmed that three species are present in our locality: F. hepatica, F. gigantica and an intermediate form which was named F. hepatogigantica n.sp. on basis of having few morphometric characters from F. hepatica (length and pattern of uterine coils) but genetically they were more related to F. gigantica.