Abstract: Genotypic analysis of 263 and 356 bp fragments of 18S rDNA obtained from Fasciola hepatica and Fasciola gigantica from Fars province using PCR-RFLP assay demonstrated that nucleotide sequences of F. hepatica from Iran differed to those reported in the other countries. PCR-RFLP bands profile using the DraI restriction enzyme differed markedly between F. hepatica and F. gigantica whereas, PCR-RFLP bands profile of F. hepatica and F. gigantica with restriction enzyme BfrI was similar together. The nucleotide sequencing results of 18S rDNA of F. hepatica and F. gigantica demonstrated 0.3% differences between Iranian F. hepatica and standard F. hepatica reported in genebank. This is the first time that molecular evidence had suggested the possible existence of an intermediate genotype of Fasciola in Iran, in addition to F. hepatica and F. gigantica as its 18S rDNA sequences were unique in that two different 18S rDNA sequences exist in the rDNA array within a single Fasciola worm. This micro heterogeneity is possibly due to sequence polymorphism among copies of the 18S rDNA array within the same worm. Based on our findings a PCR-RFLP should provide a valuable tool for the molecular identification and for studying the ecology, epidemiology and genetic structures of F. hepatica and F. gigantica especially in areas which both species co-exist, as Iran.