Abstract: Diacylglyceryl transferas (DGAT) is the key enzyme and the only rate-limiting enzyme in the synthesis of triglyceride. Identification of DGAT genes in tobacco is of great significance for creating new oil crops with potentially high value. In this study, we identified DGAT family genes from tobacco genome, and analyzed their sequence characteristics, gene structure, subcellular localization, evolutionary pattern and expression pattern. A total of 25 DGAT family proteins were obtained, with an average length of 485 amino acids, and most of them were basic proteins and hydrophilic proteins. 16 proteins located in endoplasmic reticulum, six proteins located in chloroplasts, and 3 proteins located in extracellular space. Tobacco DGAT protein contains typical WS_DGAT domain and LPLAT domain. At genetic level, 13 DGAT genes were located to 8 linkage groups. Plant DGAT family genes could be divided into four groups (DGAT1, DGAT2, DGAT3 and WSD) in phylogenetic tree. Tobacco DGAT genes belong to group DGAT2 and group WSD. The temporal and spatial expression of DGAT genes was different in differential tobacco cultivars and showed some tissue specificity, i.e., mainly expressed in senescent leaves and flower organs.