Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus(C. caninus), and Stellantchasmus falcatus(S. falcatus).Methods: Four species of heterophyid trematodes including C. caninus, S. falcatus,Haplorchis taichui and Haplorchoides sp. were amplified and the specific primer was designed based on the internal transcribed spacer 2 region. Two specific primers were used to validate the optimized PCR conditions: the specificity test and the sensitivity test.Results: Both of these specific primers confirmed the specificity through multiplex PCR reaction which generated both PCR products(231 and 137 bp) in the mixed DNA template of C. caninus and S. falcatus with no cross-reaction with other heterophyid trematodes. The optimum annealing temperature of both primers was 54–59℃. The sensitivity test used the two-fold serial dilution DNA template, which was concentrated between 10 and 0.312 5 ng/mL. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng/mL.Conclusions: The technique described here proved to be a species-specific technique and was found to be a rapid method for the diagnosis of C. caninus and S. falcatus in terms of the larval and adult stages in intermediate and/or definitive hosts in the endemic area. Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus (C. caninus), and Stellantchasmus falcatus (S. falcatus). Methods: Four species of heterophyid trematodes including C. caninus, S. falcatus, Haplorchis taichui and Haplorchoides sp. Were amplified and the specific primer was designed based on the internal transcribed spacer 2 region. Two specific primers were used to validate the optimized PCR conditions: the specificity test and the sensitivity test. Results: Both of these specific substrates confirmed the specificity through multiplex PCR reaction which generated both PCR products (231 and 137 bp) in the mixed DNA template of C. caninus and S. falcatus with no cross-reaction with other heterophyid trematodes. The optimum annealing temperature of The sensitivity test used the two-fold serial dilution DNA template, which was concentrated between 10 and 0.312 5 ng / m L. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng / mL. Conclusions: The technique described here proved to be a species-specific technique and was found to be a rapid method for the diagnosis of C. caninus and S falcatus in terms of the larval and adult stages in intermediate and / or definitive hosts in the endemic area.