Abstract: Heat shock transcription factor (Hsf) family members are widely involved in the regulation of plant development and abiotic stress responses. In the current study, 49 Hsf members were identified from the tobacco genome by bioinformatics and comparative genomics methods, all of which contained conserved oligomerization domains. The Hsf members from tobacco and Arabidopsis were categorized into 3 clades and 14 subfamilies according to the phylogenetic analysis. The number of collinear gene pairs between NtHsf and Hsf genes from dicots (Arabidopsis thaliana, tomato and grape) was more than the collinear gene pairs with the Hsf genes from monocots (rice and maize). Besides, a total of 12 NtHsf genes were predicted to have arisen from duplication events. Promoter analysis showed that there were a variety of cis-elements related to abiotic stress responses on the NtHsf promoters. The various expression patterns of NtHsf in different tissues were detected, and most NtHsf genes were induced by heat and drought stress. The results presented by this study might provide insights for the further studies on NtHsf members.