Abstract: The members of the SBP transcription factor family have been reported to play significant roles in regulating flower and fruit development as well as other biological processes. In this study, 32 SBP transcription factor family members were identified in Nicotiana tabacum with HMMER. Both Maximum-Likelihood phylogenetic tree and neighbor-joining tree were constructed and were found to possess similar topologies using the protein sequences of the SBP-domain. Results of phylogenetic analysis revealed that the SBP transcription factor family members could be classified into 8 subfamilies. The patterns of exon-intron structure and conserved domains in Arabidopsis and tobacco were consistent with the phylogenetic results. Transcriptome analysis showed that the expression patterns of NtSPLs were different in different tissue types and the expression level of NtSPLs in flower was found to be significantly higher than the other tissues. GO analysis suggested that as transcription factors, the SBP family members could be involved in a series of biological processes such as developmental regulation and defense response. The results of this study provide insight into the evolution of the SBP transcription factor family in Nicotiana tabacum and provide useful information for future research on these genes.