Comparative analysis using complete ITS2 and partial 28S rDNA sequence data revealed that cercariaeum developing in rediae in Lithoglyphus naticoides represent two different lissorchiid species. One morphotype of cercariaeum is conspecific with adult Palaeorchis incognitus from European roach, Rutilus rutilus. The other cercariaeum is attributable to the genus Asymphylodora, but the species identity is not yet determined. We also generate the first rDNA sequences for Asymphylodora progenetica based on new collections from Bithynia tentaculata from Lithuania. Phylogenetic analyses of the newly generated sequences, together with information for other lissorchiids available on GenBank, showed that all representatives of Lissorchiidae form a strongly supported clade. Three monophyletic lineages, Asymphylodora, Palaeorchis and Lissorchis, were recognized at the generic level. Karyological analysis of the chromosome set of larval P. incognitus revealed a diploid number of 2n = 20. Its karyotype with subtelocentric chromosomes prevailing can be regarded as comparatively ‘primitive’, which is consistent with the basal position of P. incognitus in the 28S tree relative to the representatives of the genus Asymphylodora. The present study adds significant new information for establishing species-specific markers for the confident characterization of different developmental stages of lissorchiid species and clarification of their life cycles.