Abstract: To investigate the effects of different tobacco varieties and climate on the expression of genes in pigment, nitrogen, starch and sugar metabolism pathway of flue-cured tobacco at mature stage, RT-PCR was used to analyze the gene expression in tobacco leaves of cv. Bina 1 and Jiucaiping 2 planted in Bijie(Guizhou) and Baofeng (Henan). The results showed that the expression levels of zeta-carotene desaturase (ZDS), chlorophyllase (CLH), glutamine synthetase (Gln1-3), nitrite reductase 3 (Nir-3), hexokinase (Hex), sucrose-6-phosphate phosphatase(SPP2)and trehalose-phosphate phosphatase(TPP)in Jiucaiping 2 planted in Bijie were higher than those in Bina 1 planted in Bijie. The results suggested that there were differences in metabolism pathway between Bina 1 and Jiucaiping 2. Nir-3 and cellulose synthase-like protein (CslE) gene expression in Bina 1 planted in Bijie expressed at a lower level and a higher level than those planted in Baofeng, respectively. The expression levels of Hex in Bina 1 and Jiucaiping 2 planted in Baofeng were higher than those planted in Bijie, and the expression of TPP in Jiucaiping 2 was lower than that planted in Bijie. This study indicated that these four genes(Nir-3, CslE, Hex and TPP) were affected mostly by climatic conditions.