Fascioloides magna is a non-native parasite in Europe which is originally a parasite of deer species in North America. After arriving to Europe, this fluke formed new host-parasite associations, and established three permanent natural foci: in Italy, Czech Republic and Danube floodplain forests. The goal of this research was to analyse cox-1 gene and to collect data on fluke diversity in Croatia and Serbia, as well as their evolutionary relations and relations with flukes collected in area of New York (Ontario), USA. After DNA isolation, PCR and sequencing we have determined 123 sequences of cox-1 genes, 58 sequences from Croatia, 55 from Serbia and 10 from the USA. In Croatia, the presence of two previously known haplotypes of the Danube foci was confirmed and four new haplotypes were discovered. The finding of new haplotypes is probably the result of the fact that samples in Croatia were collected from a larger geographical area than in previous studies, from six counties. Also, we have obtained the first data on the genetic structure of the giant liver fluke in Serbia The most common haplotype was the same as in Croatia (Ha3), and the presence of so far unidentified haplotype was found, which is common in both, Croatia and Serbia. Haplotypes obtained from USA samples were not identified in Croatia and Serbia, and of the 6 identified haplotypes only one was previously described in the literature.