Abstract: To understand the molecular mechanism underlying the establishment of leaf polarity, phenotypic analysis was performed on two long petiole mutants, M48 and M50, obtained by EMS (ethyl methanesulfonate) mutagenesis from tobacco (Nicotiana tabacum L.). Map-based cloning and comparative genomics strategies were used to isolate the mutant genes, followed by bioinformatic analysis of the candidate genes. The results showed that the long petioles of M48 and M50 were caused by the exposure of the leaf midvein, resulting from the degradation of the leaf blade from the base to the tip with different degrees. The mutant genes, Nta14g05160.1 and Nta13g05820.1, corresponding to M48 and M50, respectively, were identified as Arabidopsis REVOLUTA orthologs involved in the establishment of adaxial-abaxial polarity. Sequence similarity indicated that Nta14g05160.1 and Nta13g05820.1 originated from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid ancestors of Nicotiana tabacum L., respectively. The mutations occurred at the microRNA165 (miR165) binding site within the START domain coding sequence, suggesting that these mutations probably prevent microRNA-mediated degradation of mRNAs, resulting in increased gene transcript levels and ultimately the gain-of-function dominant mutant phenotypes.