Abstract: In order to study the genetic regulation of leaf width traits in tobacco, to locate the dominant QTL loci and promote the breeding of tobacco varieties, the recombinant inbred lines (RILs) population (Xiaohuangjin 1025×Beinhart1000-1) was genotyped using a tobacco SNP chip. The ICIM-ADD method of IciMapping 4.2 software was used to identify six QTLs related to leaf width traits in the whole genome, which were located in groups 2, 4, 9, 13, 17 and 20, respectively and could explain 2.9% to 36.8% of the phenotypic genetic variation. qMLW20-1 accounted for 36.8% of the phenotypic variation and was the major-effect QTL. A chromosome fragment substitution line which using K326 as the transmutation parent and Samsun as the dominant gene qMLW20-1 donor parent was constructed to evaluate the genetic effect of the dominant gene QTL for leaf width in tobacco. The results showed that the introduction of qMLW20-1 as the dominant gene could significantly increase leaf width, and G3 significantly increased the potassium content in tobacco leaves. The sensory quality and other important characters had no significant difference from the control, and no bad character genes were carried. The line has good breeding utilization value. The results laid a foundation for cloning and molecular improvement of the major gene of leaf width trait.