Recently, many researches for plant alkaloids, one of the largest groups of natural products, are reported because of their various pharmacological activity. This study was carried out to clone putrescine N-methyltransferase (PMT) gene which is a key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids from Burley tobacco. To induce expression of PMT gene in tobacco plant, the floral meristem was removed and then mRNA was purified from root. cDNA encoding PMT gene was isolated by RT PCR and cloned. Three different groups of clones were screened by PCR and restriction enzyme digestion analysis and were characterized. The data of these screening revealed that three types of PMT are present in Burley tobacco. Comparison of the nucleotide sequence of this three genes encoding putative PMT with those of other tobaccos revealed that two types of PMT are newly discovered from Nicotiana tabacum cv. Br21 tobacco and they were same as PMT2, PMT3 of N. tabacum cv. Xanthi.