Abstract: KNOX genes encode transcription factors with homeobox domains, which play important roles in plant morphogenesis and pattern formation. To explore the structure and functions of KNOX genes from tobacco, 18, 8 and 5 KNOX genes were identified from Nicotiana tabacum, N. sylvestris and N. tomentosiformis, respectively. In addition, their phylogenetic, gene structure, protein functional domains and gene expression patterns were analyzed. The results showed that KNOX genes from tobacco could be divided into two subclasses:NtKNOX Ⅰ and NtKNOX Ⅱ, and the two subclasses were further divided into 5 evolutionary branches. In the same evolutionary branch, the functional domains of tobacco KNOX proteins and their coding sequences were highly conserved in gene structure. Expression analysis indicated that NtKNOX Ⅱ genes in N. tabacum were expressed in multiple tissues, and the overall expression level was higher than that of NtKNOX Ⅰ genes. The high expressed genes were different in roots and leaves of N. sylvestris and N. tomentosiformis.