The metacercariae of the lung flukes, Paragonimus westermani and P. miyazakii, are of known medical importance as the pathogens causing human paragonimiasis. They are both found in the same freshwater crab species in Japan and are morphologically quite similar. The aim of the present study was to establish molecular methods for accurate discrimination between individual metacercariae of the two species. In the first step, we amplified and sequenced the second internal transcribed spacer (ITS2) region of ribosomal DNA. Searches of nucleotide databases revealed that the ITS2 sequences generated from the metacercarial DNA were identical to those previously reported for the adults of the respective species. Utilizing a nucleotide difference between the two species, we have established two PCR-based techniques; PCR-restriction fragment length polymorphism and direct PCR-amplification using species-specific primers. Both techniques showed that the individual metacercariae of the two species could be unequivocally discriminated from one another. The present results suggest that the ITS2 region is useful for species discrimination irrespective of the life cycle stages of the lung flukes. Established techniques can thus be used for epidemiological investigations of the prevalence of human lung fluke metacercariae.