The objectives of this study were to assess the response of major effect QTL to artificial selection for brown spot disease and to provide some useful information for molecular marker assisted selection (MAS) for tobacco breeding. To analyze response of major effect QTL to different artificial selection in different populations, the distribution of the all alleles at the two marker loci in the extreme populations selected from JYH/NC82 cross in different generations and a panel of 198 tobacco accessions were detected based on the major effect QTL with flanking molecular markers of J9 and J4. And there were three results: (1) the deviation of the JYH alleles at J9 loci exhibited the significance in the positive extreme populations under 5%, 10% and 20% selection intensities. The P value from X2-test for allelic frequency under different intensities (5%, 10% and 20%) were 0.015, 0.002and 0.050, respectively. (2) There was a significant increasing of the JYH allelic frequency in different generations (F3, F4, F5 and F6) derived from the cross JYH and NC82, indicating that the allele from JYH at J9 loci increase resistance to brown spot disease significantly. (3) In the panel of 198 tobacco accessions, 50 tested accessions, including highly resistant cultivars for brown spot disease such as zhongyan86, danyu2 and so on, have the same genotype with JYH cultivar. The result showed that the resistant alleles from JYH had been used widely in tobacco breeding. The major QTL associated with resistance to brown spot had been identified repeatedly and the response of major effect QTL to artificial selection for resistance to brown spot disease was evaluated. These results could provide information on MAS for improvement of resistance to brown spot disease in tobacco.