Abstract: As a plant specific transcription factor family, Dof proteins play important roles in multiple life processes of plant development. To analyze the potential roles of Dof family members in tobacco growth and development, comparative genomics, evolutionary analysis and syntenic analysis were performed to analyze potential function of NtDof members. A total of 69 Dof transcription factors were identified in the tobacco genome, and the Dof domain was highly conserved. The NtDof members were classified into 9 subfamilies based on the phylogenetic analysis. In tobacco, 16 NtDof genes were found to form 27 collinear pairs with 17 AtDof genes. The duplication events showed that 25 NtDof genes were detected to form 17 replication pairs, suggesting that replication events play a key role in the expansion of the tobacco Dof gene family. Furthermore, the expression of NtDof genes have obvious tissue specificity. NtDof05 and NtDof28 genes may be involved in root development. Tobacco Dof transcription factors were identified and analyzed in this study, and these results may provide insights for further studies on tobacco Dof family.