The bHLH transcription factors play pivotal roles in plant growth and development,production of sec-ondary metabolites and responses to various environmental stresses.Although the bHLH genes have been well studied in model plant species,a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome.In the present study,a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies.The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins.Moreover,the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs.A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome.Moreover,transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues,and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR,indicating their potential functions in the plant growth and development.Importantly,overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress.Therefore,our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.