Abstract: R2R3-MYB family is one of the largest transcription factor families in plants, and plays an important regulatory role in plant development and metabolism. To identify the biological functions and regulation mechanism of MYB12 transcription factors in tobacco, two MYB12 genes (NtMYB12a and NtMYB12b) were cloned from Nicotiana tabacum with homologous cloning strategy, gene expression patterns were analyzed by real-time quantitative PCR (qPCR), and their functions and regulation mechanisms were predicted by bioinformatic analysis. The results showed that the nucleotide sequence and deduced amino acid sequence identities between NtMYB12a and NtMYB12b were 92.91% and 87.91%, respectively. Highly conserved tobacco MYB12 genes included 4 exons and 3 introns. Phylogenetic analysis showed that NtMYB12a was evolved from Nicotiana tomentosiformis, while NtMYB12b from Nicotiana sylvestris. The expression patterns of NtMYB12a and NtMYB12b showed spatial and temporal specificity. Under topping, salinity stress, drought, darkness and phosphate starvation treatments, the expression levels of NtMYB12a and NtMYB12b were significantly different. The expression level of NtMYB12a in tobacco leaves presented an increase tendency after topping. Salinity stress, darkness and phosphate starvation significantly down-regulated the expression level of NtMYB12a, in comparison with no effect of drought. The expression levels of NtMYB12b were up-regulated under all treatments. The results indicated that NtMYB12 might play an important regulatory role in responding to abiotic stress.