Abstract: To explore the molecular mechanism of water-soil two-stage seedling cultivation method against low temperature stress, bioinformatic methods were used to analyze the evolutionary relationship, gene structure, conserved domains and cis-acting elements in promoter of CBF (C-repeat binding factor) gene family in tobacco. Furthermore, qRT-PCR was used to analyze the responsive models of NtCBF gene family, CBF-regulon related genes and non-CBF-regulon related genes to low temperature under two seedling cultivation methods, conventional floating seedling cultivation and water-soil two-stage seedling cultivation. The results showed that the evolutionary relationship between NtCBF15-NtCBF21 gene and CBF5 or CBF6 of Arabidopsis thaliana was close, and that between NtCBF1-NtCBF14 genes and CBF1-CBF4 genes of Arabidopsis thaliana was close. All members of NtCBF gene family had conserved protein domains of motif 1/2/4/5, indicating that the family was relatively conserved during evolution. Among them, promoter regions of eight genes (NtCBF3, NtCBF4, NtCBF5, NtCBF6, NtCBF10, NtCBF13, NtCBF14 and NtCBF18) had low temperature response elements. The expression levels of NtCBF5, NtCBF12 and NtCBF13 of NtCBF gene family and NtRD29A, NtRD29B and NtGOLS3 of the CBF-regulon related genes were significantly increased in water-soil two-stage seedling after the cold treatment. The evidence suggested that NtCBF5, NtCBF12, NtCBF13 and their downstream regulatory genes NtRD29A, NtRD29B and NtGOLS3 might play important roles in the resistance of seedlings cultured by the two-stage method to low temperature stress.