A new species and three known species of family Didymozoidae Monticelli, 1888 from marine fishes in Japan, with records of their morphological characteristics and DNA markers.

The Trematoda family Didymozoidae comprises over 250 species that typically use marine fishes as their definitive hosts. Species identification of adult didymozoids based solely on morphology is often problematic owing to the difficulty of observing their detailed internal organs. Although molecular analyses are generally essential for trematode taxonomy, reference sequences (e.g. 28S rDNA, ITS, and COI) for morphologically identified adult didymozoids remain scarce. In this study, adult didymozoids were collected from five marine fish species in Japan between 2018 and 2025. Morphological observations revealed four species, including Annulocystis carnata n. sp. from Euthynnus affinis. The new species is distinguished from the other members of the genus Annulocystis by its robust forebody, subglobular oral sucker, and oval testes. Three known species, Annulocystis auxis (from Auxis thazard), Gonapodasmius squamatus (from Mola mola), and Nematobothrium filiforme (from Scomber spp.), are redescribed and linked to novel molecular markers. The 28S rDNA sequence of Annulocystis carnata n. sp. was identical to that of a previously reported Annulocystis cf. auxis from Australia, suggesting that they are conspecific. Our molecular analyses revealed that some metacercariae reported from cnidarian jellyfishes in a past study were conspecific with N. filiforme. These results suggest the critical need for molecular markers in didymozoid identification. Our 28S rDNA-based phylogenetic analysis indicates that the currently accepted macrotaxonomy of the family may require revision for some genera and subfamilies.