Abstract: MADS-box family genes play an important role as transcription factors in plant developments, especially in the development of floral organ, and florescence. Early flowering in tobacco agricultural production is associated with MADS-box family genes. In order to isolate new MADS-box family genes from tobacco, degenerate primers were designed according to conserved domain of various plant MADS-box family genes, and 27 cDNA fragments were amplified from the young shoot tip of tobacco variety NC82 using RT-PCR technology, fragment lengths were between 122 bp and 146 bp. By using Blast in NCBI, 16 among these 27 cDNA fragments’ deduced amino acid sequences contained MADS_MEF2_like region. Multiple sequence alignment analysis indicated that these 16 deduced amino acid sequences had high homology with MADS-box family genes of Arabidopsis and other species, the average identity could reach 76%. Meanwhile, the 16 fragments contained 15 conserved amino acid motifs, of which 7 motifs involved in the interaction between protein and DNA, 4 motifs with protein dimerization function. Phylogenetic analysis shows these conserved MADS-box family gene fragments could be classified under different MADS-box subfamilies (FLC, STMADS11, TM3, AGL15, AG, AGL2, AGL6, DEF/GLO, and AGL12). FLC, STMADS11, TM3 and AGL6 were associated with flowering. These results indicated that there might be several MADS-box family genes involved in low temperature response and early flowering of cold-sensitive tobacco NC82.