Background and Objective: Fascioliasis is an important zoonotic disease that causes several health problems and economical losses in different parts of Iran including Zanjan. Fasciola hepatica and F. gigantica are recognized as causative agents of the disease. The differential diagnosis between these two species is very important for planning and control of infection. This study was designed to identify the Fasciola species by molecular methods in Zanjan (Iran). Methods and Materials: A number of 535 adult Fasciola worms were collected from the natural infected livers of cattles and sheep in local slaughterhouse. Living flukes were washed extensively in PBS at 37 OC and then anterior half of adult worms were stored at -20 OC in 70% ethanol. Total genomic DNA was extracted from individual flukes by modified phenol-chloroform method. Nucleotide polymorphism of ITS2 fragment of rDNA was investigated using PCR-RFLP assay and sequencing technique. Results: The results of PCR-RFLP and comparison of ITS2 sequences with the BLAST GenBank database clarified that all specimens were F. hepatica. The obtained sequences are available in the GenBank, with accession numbers EU391412 to EU391424. Conclusion: The results of this study showed no evidence of F. gigantica infection in sheep and cattles in Zanjan as all of the isolates were found to be F. hepatica. Key words: Fasciola, Liver flukes, rDNA, ITS2, PCR-RFLP