Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the present study, samples identified morphologically as Fasciola hepatica from sheep and cattle from different geographical locations of Tunisia and Algeria were genetically characterised by sequences of the first (ITS-1), the 5.8S and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) and mitochondrial Cytochrome c Oxidase subunit I (COI) gene. Comparison of the ITS and COI sequences of the North African samples with sequences of Fasciola spp. from GenBank confirmed that all samples from Tunisia and Algeria samples belong to a single species, namely F. hepatica. Several specimens from Tunisia and Algeria showed a substitution C/T in position 859 in the ITS-2 sequences, previously reported from Spain, suggesting that the above mentioned variant may have a common origin and spread recently throughout the three countries because of movement of infected animals. This is the first molecular characterization of F. hepatica in North Africa which provides a foundation for further studies on Fasciola spp. in Tunisia and Algeria.