Powdery mildew caused by Erysiphe cichoracearum is a major disease of tobacco affecting a wide area including Asia. The Japanese domestic air-cured tobacco cultivar Kokubu (syn. Kou-fan) exhibits extremely high resistance to powdery mildew by recessive alleles at two loci. This resistance has been introduced into several cultivars. Although the genes responsible for this recessive resistance are not identified, the loss-of-function mutation in the Mildew resistance locus O (MLO) gene confers broad-spectrum resistance to powdery mildew in various plant species. To elucidate the molecular mechanism of powdery mildew resistance of Kokubu, transcripts and genomic sequences of tobacco MLO orthologues were compared between powdery mildew resistant and susceptible cultivars. The tobacco genome included two MLO orthologues (NtMLO1 and NtMLO2) corresponding to powdery mildew resistance. However, transcripts of both NtMLO genes of powdery mildew resistant cultivars included partial deletions or insertions. In DNA sequence alignment analysis, the dinucleotide substitution at the 3’ end of intron 7 in NtMLO1 gene and the dinucleotide deletion at the 5’ end of intron 6 in NtMLO2 gene were found in powdery mildew resistant cultivars. These mutations triggered the splicing alterations in NtMLO transcripts in powdery mildew resistant cultivars. Transgenic Kokubu expressing wild-type NtMLO1 or NtMLO2 exhibited severe disease symptoms, as did susceptible cultivars. These results demonstrate that powdery mildew resistance in Kokubu results from the splicing mutations of two NtMLO genes and the subsequent inhibition of functional MLO protein synthesis. Using the differences of nucleotide sequence of NtMLO genes, we developed DNA markers to detect the powdery mildew resistant or susceptible genes. These markers are expected to simplify the breeding of powdery mildew resistant cultivars and to obviate the use of test crosses for genotype determination.