Abstract: In silicon cloning, three distinct defensin genes (NtDEF1, NtDEF2 and NtDEF3) were obtained successfully from tobacco cultivar K326 by using pepper CaDEF1 gene sequence (AF442388) to be probe. Sequence analysis showed that the in silicon cloned cDNAs had a complete open reading frame (ORF) and encoded 78, 77 and 76 amino acid residues, respectively, including a strictly conserved γ-thionin domain of eight cysteines which is characteristic in plant defensins. Multiple alignment analysis based on the deduced amino acid sequences of defensin genes from different pIants indicated that NtDEF1, NtDEF2 and NtDEF3 showed high similarity with 50%-87% identities to the defensins from Capsicum annuum. However, with 33%-40% identities to the defensins previously reported from Nicotiana, the evolutionary analysis showed that NtDEF1, NtDEF2 and NtDEF3 were very far related to them. In conclusion, this is the first time that the NtDEF1, NtDEF2 and NtDEF3 genes have been reported in tobacco.