Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, the trematodes of the family Opisthorchiidae, are important human parasites. Two previous studies (Kang et al. Parasitol Int 57:191-197, 2008; Katokhin et al. Dokl Biochem Biophys 421:214-217, 2008) have provided evidence using ribosomal and mitochondrial sequences that O. viverrini, O. felineus, and C. sinensis are closely related. We developed a novel nuclear marker, Pm-int9, which included the ninth intron of the paramyosin gene and flanking exon sequences. Samples of O. felineus from four localities of West Siberia, C. sinensis from the Russian Far East, and O. viverrini from Thailand were genotyped by Pm-int9. Little variation was detected in exon sequences, however, intron sequences turned out to be more variable than ribosomal internal transcribed spacers. We can conclude that Pm-int9 is valuable for interspecific variation studies. Phylogenetic analysis based on Pm-int9 revealed that O. viverrini and C. sinensis were closer to each other than either of them to O. felineus, supporting the opinion that C. sinensis should be considered the sister species of Opisthorchis spp.