Abstract: Chestnut blight caused by Cyphonectria parasitica is the main disease in Castanea mollissima cultivation area. According to the nucleotide sequence of internal transcribed spacer regions(ITS)of the ribosomal gene of Cryphonectria in GenBank, a couple of primers CP1/CP2 for C. parasitica were developed. By random amplification polymorphism technology, we identified a specific RAPD fragment of C. parasitica from all the strains tested. After recycling and purification, the specific fragment was cloned and sequenced. The sequence was used to design two specific primers SC1/SC2 and converted RAPD marker to SCAR marker successfully. Combining the primers CP1/CP2 and SC1/SC2, a duplex PCR method for detecting C. parasitica had been established. The result indicated that the duplex PCR could amplify two unique fragments of 285 bp and 389 bp in size from C. parasitica,but other strains tested didn't present any fragments. Sensitivity testing showed that the detection limit was 300 fg/μL for genomic DNA. The duplex PCR method also successfully detected C. parasitica from infected chestnut tissues. Our results suggested that the duplex PCR method would have great significance for accurate identification and detection of C. parasitica.