Mature worms of Stephanoprora amurensis sp. nov. were obtained in an experimental study of its life cycle. In the Russian southern Far East, this trematode circulates using freshwater snails Parajuga subtegulata, freshwater fish and birds as the first, second intermediate and final hosts, respectively. Stephanoprora amurensis sp. nov. differs from the well-known representatives of Stephanoprora in a number of morphometric indicators of the developmental stages. The validity of the species was also confirmed by nuclear and mitochondrial DNA markers. In addition, new genetic data were obtained for Echinochasmus suifunensis and Echinochasmus milvi. An analysis of phylogenetic relationships within Echinochasmidae based on the 28S rRNA gene and ITS2 region identified two clusters, one of which combines species of Echinochasmus with 20–22 collar spines and short-tailed cercariae, and the other which includes Stephanoprora spp. and a number of representatives of Echinochasmus with 24 collar spines and long-tailed cercariae. The results of phylogenetic analysis based on ITS2 data show interfamily level of differences between the two clusters and intergeneric differentiation between the three subclusters uniting the species of Stephanoprora and Echinochasmus.