Abstract: Background and Objective: Fasciola hepatica is a zoonotic liver fluke infects wide range of hosts (all ruminant animals, rabbits, horses and human). It is commonly prevalent where the intermediate snail host is present. The main objective of this study was to provide molecular characterization of F. hepatica using ribosomal DNA cluster (rDNA) of more conserved Internal Transcribed Spacer (ITS) regions and mitochondrial cytochrome oxidase subunit I (COI) gene. Materials and Methods: Adult fluke samples were collected from livers of infected cattle hosts. All flukes were subjected to DNA extraction for polymerase chain reaction. Results: A 421 bp fragment of COI and 603 bp ITS2 genes were amplified, purified and sequenced. The obtained sequence was compared to the corresponding sequences available in the GenBank. Phylogenetically, F. hepatica resembles closely the other members of family Fasciolidae, showing considerable, expectation value in the alignment. Conclusion: This study corroborate that the mitochondrial COI and ITS2 sequences could be used as species specific markers for characterization of F. hepatica.