Echinostome metacercariae were investigated in freshwater snails from 26 districts in 7 provinces of upper northern Thailand. The species identification was carried out based on the morphologies of the metacercariae and adult flukes harvested from experimental hamsters, and on nucleotide sequences of internal transcribed spacer 2 (ITS2) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) genes. Twenty-four out of 26 districts were found to be infected with echinostome metacercariae in freshwater snails with the prevalence of 40.4%. The metacercariae were found in all 6 species of snails, including Filopaludina martensi martensi (21.9%), Filopaludina doliaris (50.8%), F. sumatrensis polygramma (61.3%), Bithynia siamensis siamensis (14.5%), Bithynia pulchella (38.0%), and Anenthome helena (4.9%). The echinostome metacercariae found in these snails were identified as Echinostoma revolutum (37-collar-spined) and Echinostoma macrorchis (45-collar-spined) morphologically and molecularly. The 2-week-old adult flukes of E. revolutum revealed unique features of the cirrus sac extending to middle of the ventral sucker and smooth testes. E. macrorchis adults revealed the cirrus sac close to the right lateral margin of the ventral sucker and 2 large and elliptical testes with slight indentations and pointed posterior end of the posterior testis. The ITS2 and nad1 sequences confirmed the species identification of E. revolutum, and the sequences of E. macrorchis have been deposited for the first time in Gen-Bank. The presence of the life cycle of E. macrorchis is a new record in Thailand and the snail F. doliaris as their second intermediate host seems to be new among the literature.
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