Abstract: It has been demonstrated that Ax1/Ax2* and Dx5 are normally associated with wheat flour superior end-use quality, especially dough strength. This paper was designed to improve the molecular marker identification efficiency by the duplex allele-specific polymerase chain reaction (Duplex AS-PCR). In distinguishing the high molecular weight glutenin subunit, the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) proved that the established duplex AS-PCR was credible. The genes of Ax1/Ax2* and Dx5 could be identified simultaneously in this polymerase chain reaction system. The presence of 1500 bp and 478 bp bands showed Ax1/Ax2* and Dx5 genes, respectively. A total of 21 wheat cultivars abroad were tested by the duplex allele-specific PCR-based assay and the frequencies of Ax1/Ax2* and Dx5 were 33.3% and 52.4%, respectively. In conclusion, the amplification reaction system was stable, and it could be used to simultaneously identify Ax1/Ax2* and Dx5 subunits, so it would be very helpful for marker-assisted selection of new wheat varieties with high quality.