Cold stress is a major adverse environmental factor that affects plant growth, development, productivity and quality. In the present study, comparative genome-wide transcriptome analysis on two tobacco (Nicotiana tobacum L.) cultivars, cold-tolerant NC567 and cold-sensitive Taiyan8, was performed using RNA-seq technology. After the first assembly, total length of unigenes is from 101,308,644 to 123,781,795 bp, the N50 length is from 1357 to 1475 bp, and 152,688 unigenes in NC567 and 144,160 unigenes in Taiyan8 were identified, respectively. Functional classification of cold-responsive (COR) genes showed that the genes involved in cell wall metabolism, transcription factors, ubiquitin-proteasome system (UPS) and signaling are over-represented, and the COR genes are specifically induced during cold stress in NC567. Pathway analysis revealed the significant enrichment of the COR genes in plant circadian clock. Taken together, the present study suggested the positive roles of the highly induced expression of the COR genes and the conserved mechanism of circadian clock related genes in tobacco response to cold stress, and provided some valuable genes for crop improvement to cope with cold stress. (C) 2015 Elsevier Inc. All rights reserved.