Isolates of Dicrocoelium dendriticum (n=150) from sheep and cattle bred in southern Italy and isolates (n=5) of D. hospes from a Bos indicus from Senegal were characterized genetically. The 28S region and the second internal transcribed spacer (ITS-2) plus flanking 5.8S and 28S sequences (ITS-2+) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction and sequenced from individual flukes. Regarding the 28S rDNA, sequences of 568 and 581 bp were obtained for D. dendriticum and D. hospes, respectively. No intraspecific variation was observed between the 28S rDNA of all the D. dendriticum specimens studied and the D. dendriticum 28S rDNA sequence present in GenBank. However, intraspecific variation was observed in the 28S rDNA of the D. hospes specimens compared to the sequence present in GenBank. Regarding the ITS2+ rDNA, sequences of 402 and 428 bp were obtained for D. dendriticum and D. hospes, respectively; both sequences were deposited in GenBank. Variations intra- and interpopulation were observed for D. dendriticum, whereas 100% identity was observed in all the ITS2+ sequences of D. hospes. With respect to the interspecific variations, the ITS-2+ of D. dendriticum and D. hospes differed in 33 positions. The findings of the present study showed an ITS2+ sequence variability (8.2-8.5%) between D. dendriticum and D. hospes, thus demonstrating the utility of this sequence to discriminate the two species.