Cinnamic acid -4- hydroxylase (C4H) and ρ-coumaric acid -4- ligase (4CL) are the key enzymes in phenylpropanoid metabolic pathway, whose polyphenol products is corresponding to the quality of tobaccos. Here, cDNAs of these two enzymes were obtained using homology-based cloning from variety honghua dajinyuan (HD) and variety K326, whose quantity of polyphenol products differed significantly and their expression profiles were analyzed by Real time qRT-PCR. The results showed that Ntc4h and Nt4cl both have two homologous genes. The ORF length of Ntc4h1, Ntc4h2, Nt4cl1 and Nt4cl2 was 1518 bp, 1518 bp, 1644 bp and 1629 bp, respectively. According to the analysis, Ntc4h1, Nt4cl1 and Ntc4h2 have shown differences in these two varieties on coding sequence. Real time qRT-PCR analysis showed that all the genes expressed in a spatial and temporal manner and the expression profiles differed in two varieties. The genes all expressed in root, stem, leaf, flower and sepal with the expression of xylem and phloem were higher than others. The expression in fast growing stage and technical maturity stage was higher than the others with the highest in technical maturity stage.