Abstract: In order to improve the efficiency of genotype selection of recessive resistance genes (susceptibility genes) NtMLO1 (M1) and NtMLO2 (M2) to tobacco powdery mildew in molecular marker-assisted selection breeding, on the basis of the reported nucleotide sequences of M1 and M2 mutant genes, four pairs of primers were designed to develop two multiplex polymerase chain reaction systems, which were separately used to amplify wild-type or mutant alleles of M1 and M2 simultaneously. The results showed that the specific fragments amplified by the two established duplex PCR systems were identical with those amplified by the monoplex PCR systems. Mutant homozygote produced specific fragments of 191 and 306 bp only in mutant allele-specific multiplex PCR systems, and wild-type homozygote produced specific fragments of 437 and 652 bp only in wild-type allele-specific multiplex PCR systems, however heterozygote produced specific fragments in both multiplex PCR systems. These systems can be used to accurately detect the genotypes of M1 and M2 in backcross or selfed progenies during backcrossing via one or two multiplex PCR amplification, so as to speed up the breeding process of new disease-resistant cultivars.