Abstract: To elucidate the molecular mechanism underlying petiole formation in Nicotiana tabacum L., near-isogenic lines were constructed to precisely evaluate the genetic effect of a dominant mutant gene controlling the short petiole phenotype in tobacco. Fine-mapping was performed by enlarging the segregation population and developing new molecular markers based on whole genome sequencing. Bioinformatics prediction and analysis were subsequently conducted to identify candidate gene. The results demonstrated that the dominant mutant gene primarily regulated the formation of short petiole in the mutant, without causing notable effect on other leaf traits. The gene was located to a 210-Kb interval on Chrosome 7 of NtaSR1, where a KNOX Ⅰ gene, Nta07g20040.1, was identified as the candidate gene. No sequence changes were detected in the coding region of Nta07g20040.1 in the mutant; however, four SNP mutations consistent with the ethyl methanesulfonate (EMS) mutagenesis pattern were found tens of kilobases upstream and downstream of the coding sequence. These mutations were absent in the wild-type tobacco lacking petioles and in its ancestral species N. sylvestris. It is hypothesized that these SNPs likely have induced a gain-of-function dominant mutation in Nta07g20040.1, leading to the degradation of leaf tissue at the base of the midrib and subsequent petiole formation. Nta07g20040.1 has a highly homologous gene, Nta08g17660.1, in N tabacum, and sequence similarity analyses indicated that the two genes originated from N. sylvestris and N. tomentosiformis, the diploid ancestors of N. tabacum, respectively.