Abstract: miRNA169 is a key factor involved in early flowering at low temperatures. In order to understand the expression and regulation mechanism of miRNA169 gene family in tobacco at low temperatures and to breed new tobacco varieties with resistance to low temperatures and early flowering, tobacco miRNA169 data in miRBase were analyzed. The structure and function of miRNA169 and their promoters in tobacco were predicted. The results showed that 19 miRNA169 genes were identified in tobacco genomes. They were distributed on chr01, chr08 and chr09 chromosomes. miRNA169 was found to cluster together in one chromosome. Except for pre-miRNA169t, the other pre-miRNA169 had high homology. LTR elements, corresponding to ABA and JA and involved in low temperature stress, were found on all promoters of miRNA169 in tobacco. Therefore, it was speculated that the response process of miRNA169 to low temperature stress might be the result of the interaction of LTR, ABA and JA pathways in tobacco.