Many freshwater snail species of the family Lymnaeidae are known to act as intermediate hosts of Fasciola hepatica, the causative agent of fasciolosis, a parasitic disease of great veterinary and medical importance. Morphological identification of lymnaeid species is based on characteristics of shell, reproductive and renal systems. However, such identification is challenging due to interspecific similarity, which is particularly evident among the species Galba viator, G. cubensis, and G. truncatula. To overcome these difficulties, we used molecular markers and a morphological approach applied to specimens from Brazil to compare the data obtained with type specimens, topotypes, and other lymnaeid species. We used PCR-RFLP with MboII, HpaII, and AluI enzymes directed to the ITS2-rDNA region, which allowed differentiation among Pseudosuccinea columella, Pectinidens diaphanus, G. viator, G. cubensis, and G. truncatula when morphological characterization is inconclusive. These results allowed us to report for the first time the occurrence of G. cubensis and G. truncatula in the state of Minas Gerais, Brazil. We also performed phylogenetic analyses using 16S mtDNA sequences, which corroborated the PCR-RFLP results. The molecular techniques used in this study aimed to support the morphological identification of Lymnaeidae populations from Brazil and have proven to be a helpful tool when morphological analysis does not support confident identification at the species level.