Fasciola hepatica is a trematode parasite that affects mammals, including humans. In Brazil, fascioliasis, a disease caused by the parasite, is of great importance. The disorder affects the welfare of the Brazilian population through impairing the agricultural production of cattle, where the disease causes weight loss as a result of liver damage. This study aimed to evaluate the genetic diversity of F. hepatica throughout Southern Brazil to determine its geographic origin and estimate the colonization route of the parasite. To accomplish these aims, flukes were collected from slaughterhouses in three endemic areas of Rio Grande do Sul and Paraná states. DNA was isolated using the phenol-chloroform protocol from single flukes and two mitochondrial genes, cytochrome oxidase subunit I (COI) and nicotinamide dehydrogenase subunit 1 (Nad1), were amplified and sequenced. Ten haplotypes of COI were found from 75 isolated parasites and the total haplotype and nucleotide diversity observed were 0.475 and 0.002, respectively. Using the Nad1 gene, we found 24 haplotypes from 79 samples, resulting in haplotype and nucleotide diversity values of 0.756 and 0.004, respectively. An analysis of molecular variance showed that 57.4% and 77.5% of variation was within populations (FST), while 9.0 and 36.8% of variation was among groups (FCT) when considering COI and Nad1 genes, respectively. For COI, the fixation index values of 0.425 and 0.368 were obtained for FST and FCT, respectively, while analysis of Nad1 0.225 and 0.089 index values were obtained for FST and FCT, respectively. We have determined that F. hepatica found in the two distinct areas originated from several geographical regions, since we found haplotypes that were shared with at least three different continents. These data are in accordance with the recent colonization of Brazil, and the recent import of cattle from South American, European and, possibly, some African countries. The observed FST and FCT values for COI and Nad1 genes of F. hepatica may be a result of limited movement of animals within states and support the lack of geographical structure of the parasite in Brazil, which are in agreement with the observed cattle production systems in this region.