Background: CB-1 and K326 are closely related tobacco cultivars; however,their cold tolerance capacities are different.K326 is much more cold tolerant than CB-1.Results: We studied the transcriptomes and metabolomes of CB-1 and K326 leaf samples treated with cold stress.Totally,we have identified 14,590 differentially expressed genes(DEGs)in CB-1 and 14,605 DEGs in K326; there was also 200 differentially expressed metabolites in CB-1 and 194 in K326.Moreover,there were many overlapping genes(around 50%)that were cold-responsive in both plant cultivars,although there were also many differences in the cold responsive genes between the two cultivars.Importantly,for most of the overlapping cold responsive genes,the extent of the changes in expression were typically much more pronounced in K326 than in CB-1,which may help explain the superior cold tolerance of K326.Similar results were found in the metabolome analysis,particularly with the analysis of primary metabolites,including amino acids,organic acids,and sugars.The large number of specific responsive genes and metabolites highlight the complex regulatory mechanisms associated with cold stress in tobacco.In addition,our work implies that the energy metabolism and hormones may function distinctly between CB-1 and K326.Conclusions: Differences in gene expression and metabolite levels following cold stress treatment seem likely to have contributed to the observed difference in the cold tolerance phenotype of these two tobacco cultivars.