To identify the sex of Schistosoma japonicum cercaria, single sex cercariae were prepared. Monosexual miracidia were used to infect single parasite-negative snail for breeding positive snails being able to shed single sex cercaria. The single-sex cercariae released from each positive snail were collected for sex identification using the sequence characterized amplified region-PCR (SCAR-PCR) method. To confirm the sex of cercariae obtained, single-sex cercariae from the same snail were used to infect mice, and the adult worms were harvested on 21 d post-infection for verifying the worm gender morphologically, and examining the accuracy of the PCR method for detecting DNA of one single cercaria. The PCR results indicated that the female cercariae presented a specific band at 153 bp, showing a concordance with the findings of infected mouse experiment. The minimum DNA amount detectable in the PCR assay was 16.5 ng, equivalent to the DNA amount of one cercaria. To verify the accuracy of the PCR method, 40 female and 40 male sex-known cercariae verified morphologically on their derived adult worms were examined using the PCR method, and found 37 females and 40 males respectively from the sex-known samples, indicative of a sex-discriminating accuracy of 92.5% (37/40) for female and 100% (40/40) for male cercariae of S. japonicum. The results demonstrated that the cercaria sex could be differentiated using the SCAR-PCR method.