Abstract: To explore the molecular mechanism of carbon metabolism in tobacco leaves after harvest, the flue-cured tobacco varieties "Yuyan 12" and "Qinyan 96" were taken as the sample materials to determine the changes of starch, total sugar, and reducing sugar contents, and the expression levels of starch and sucrose metabolism related genes in tobacco leaves after harvest were analyzed by real-time fluorescence quantitative PCR. The results indicated that the starch content of "Qinyan 96" was significantly higher than that of "Yuyan 12". Starch content decreased gradually after harvested for 0-30 h, while total sugar and reducing sugar gradually increased, and sugar content increased rapidly after 12 h. The expression of NtAGPS3, NtGBSS1, NtSS1, NtSS2, NtSBE1, NtSBE2, NtISO2, NtISO3, NtSP, NtSPSA, NtSPSB, NtSPSC, NtSPP1, NtSPP2, NtSuS and NtVIN1 in fresh tobacco leaves of "Qinyan 96" was significantly higher than that of "Yuyan 12", while the expression of Ntα-amylase and Ntβ-amylase was significantly lower than that of "Yuyan 12". The expression levels of NtAGPS1, NtAGPS3 and NtGBSS1 decreased rapidly after harvested for 0-30 h; the expression levels of amylopectin synthesis related genes showed disordered increasing and decreasing; the expression of NtSR1, Ntα-amylase, Ntβ-amylase, NtSP, NtSPSA, NtSPSB, NtSPSC, NtSPP1, NtSPP2, NtSuS and NtVIN1 showed the trend of ascending first then descending, or increasing gradually. The expression levels of NtAGPS1 and NtGBSS1 were significantly correlated to the changes of starch and sugar contents. These results suggested that there are some differences in starch and sugar metabolism between "Yuyan 12" and "Qinyan 96", and NtAGPS1 and NtGBSS1 might be the key genes regulating the carbon metabolism in tobacco leaves after harvest.