Abstract: To breed new tobacco varieties with no or less axillary buds, based on CRISPR/Cas9 system, the DNA sequence of NtLS was chosen as the target site for designing two CRISPR/Cas9 vectors (cLS1 and cLS2). Agrobacterium tumefaciens-mediated transformation of cv. K326 was used and twenty kanamycin-resistance tobacco plants (T₀ generation) were obtained. Ten positively transformed tobacco plants were analyzed by PCR amplification, sequencing and alignment, and some mutations of NtLS protein were detected in two plants. These research results provide useful mutants to study the molecular mechanism of axillary bud formation and create genetic materials for new flue-cured tobacco varieties with no or less axillary buds.