The present study was performed to record new definitive hosts of Isthmiophora hortensis, and to describe morphological characteristics derived from a variety of worm samples for clarification of its taxonomic validity. Morphological characteristics with dimensions were observed in worm samples (n=21) from naturally infected wild animals, including a raccoon dog Nyctereutes procyonoides from Gimhae-si (City), Gyeongsangnam-do, stray cats and a striped field mouse from several localities, and a wild boar Sus scrofa, from Gurye-gun (County), Jeollanam-do. In addition, adult flukes (n=45) recovered in albino rats experimentally infected with the metacercariae from a freshwater fish species were also subjected to morphological studies. The mean ratios of the body length (BL) to body width (BW) were 5.86 and 5.76 in worms from wild animals and experimental rats, respectively. Those of the ventral sucker to oral sucker were 2.92 and 3.01 in worms from 2 groups. The mean percentages of the hindbody length (HBL) to BL were 42.1 and 41.2 in 2 groups. Those of uterine fields to BL were 9.8 and 12.2 in the 2 worm groups. By the present study, the 2 species of wild animals, the raccoon dog and wild boar, have been added as new definitive hosts for I. hortensis. The morphological characteristics of adult flukes derived from a variety of host source were redescribed to support the taxonomic validity of this echinostome species.