DNA barcoding is a technique to identity species by using standardized DNA sequences. In the present study, a total of 105 samples, representing 30 Parnassia species were collected to test the effectiveness of four proposed DNA barcodes (rbcL, matK, trnH-psbA and ITS) for species identification. Our results demonstrated that all four candidate DNA markers have a maximum level of primer universality and sequencing success. As single DNA marker, ITS region provided the highest species resolution with 86.7%, followed by trnH-psbA with 73.3%. The combination of the core barcode, matK + rbcL, gave the lowest species identification success (63.3%) among any combination of multiple markers, and was found unsuitable as DNA barcode for Parnassia. The combination of ITS + trnH-psbA achieved the highest species discrimination with 90.0% resolution (27 out of 30 sampled species), equal to the four-marker combination; ITS combined with matK, rbcL or matK + rbcL did not improve species discrimination. Therefore, matK and rbcL should not be used as DNA barcode for species identification of Parnassia. Based on the overall performance, the combination of ITS + trnH-psbA, is proposed as the most suitable DNA barcode for identifying Parnassia species. DNA barcoding is a useful technique to providing a reliable and effective mean for the discrimination of Parnassia species, and in combination with morphology based taxonomy can be a robust approach for tackling taxonomically complex groups. In the light of our findings, we found among the three species not identified a possible cryptic speciation event in Parnassia.