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Rice blast is one of the important diseases in major rice producing areas in China. The main blast resistance genes Pi-ta and Pi-b showed wide and durable resistance spectrum in many rice growing areas. In this study, two sets of multiple PCR system primed by functional markers were established to identify Pi-ta and Pi-b, using 22 rice varieties with known alleles on both Pi-ta and Pi-b loci. The PCR system I could detect the resistance alleles on Pi-ta and Pi-b loci, while the PCR system II could detect the susceptible alleles simultaneously. The effectiveness of the two PCR systems was validated in 336 japonica breeding lines. The result was highly consistent with that of molecular detection using conventional single marker. Therefore, the two multiple PCR systems developed are stable, reliable and time-saving, and can serve as a rapid and efficient method to identify Pi-ta and Pi-b genes in bulky screening and marker-assistant selection in rice aiming at blast resistance breeding.