Background: Fasciola hepatica as an important parasite affects health of humans and animals in some tropical and subtropical areas of the world, including Iran. Little is known about the molecular diversity of Fasciola in Equidae. Therefore, this study aimed to characterize the genetic polymorphisms among parasites. Methods: Eight adult Fasciola spp. isolates were collected from a working donkey after necropsy in Shiraz, southwestern Iran, in 2018. Primarily, various parameters were measured morphologically. Subsequently, DNA was extracted from each fluke and molecular markers of cytochrome C oxidase (cox1) and NADH dehydrogenase 1(nad1) from individual Fasciola isolates were amplified using PCR assay and sequence data were employed for molecular and phylogenetic analysis. Genetic diversity between isolates was evaluated by comparing the sequences of these two mitochondrial regions. Results: Based on the morphological and analyzed mitochondrial sequences, all of eight donkey isolates (100%) were identified as F. hepatica. Moreover, nine and five nucleotide polymorphisms were identified in the cox1and nad1 region sequences, respectively. Conclusion: Accordingly, phylogenetic data revealed five and four haplotypes among donkey isolates based on the cox1and nad1 markers. Similarly, some of these haplotypes have been previously reported from different host species in Iran as well as all around the world.