BACKGROUND: Dicrocoeliasis is one of the commonest parasitic diseases of the bile ducts and gallbladder in a wide range of mammals including ruminants and sometimes human being. The disease is really important in medicine, economy, and veterinary medicine. Annually, slaughter house loses a huge amount because of losing a great deal of valuable proteins in people daily diets. In order to bring the disease under control, morphological and molecular analysis of parasite in endemic districts is essential. OBJECTIVES: The present study was conducted to determine the morphological as well as molecular characterization of cattle, sheep and goats isolated from Dicrocoelium by applying ND1 genetic marker in the Markazi province, Iran. METHODS: In this cross-sectional study 480 fresh adult worms were collected from livers of 120 cattle, sheep and goats slaughtered in abattoirs in Markazi province. To diagnose the species of parasite, morphometric indices of mature worms were calculated based on standard parameters. Then DNA of 60 isolates with different morphometric characteristics was extracted and PCR reaction was performed for a part of ND1 (mtDNA). PCR was purified and its sequence was defined, the percentage of genetic similarity was compared to cases registered by GenBank and the exact species of parasite was recognized. RESULTS: The morphometric analysis in all isolates was as follows: testicles were sequential, the length and the width of the worm for all cattle, sheep and goat isolates were 7994±967µm, 6844±100µm, 6570±110µm (P<0.0001) and 1649±339µm, 1490±221µm and 1430±252µm (P<0.0001) respectively. The proportion of the length to the width was 4.87±0.641, 4.58±0.625, and 4.64±0.622 respectively. All the results mentioned above confirmed Dicrocoelium dendriticum in the hosts of the district under investigation. The analysis of the gel electrophoresis in all isolates showed the existence of band 200pb.The percentage of genetic similarity to the registered items, cases were determined by the Gen bank between 97 and 99 percent. CONCLUSIONS: Molecular identification and morphometric assays clearly showed that D. dendriticum is the only agent of Dicrocoeliasis among cattle, sheep and goats in the Marakazi province, Iran. Molecular diagnosis of parasite by applying genetic marker of the nucleus is recommended.