Tobacco is harvested after plant flowering, when the leaf senescence is more or less visible depending on cultivars. Before the drying phase, current practices allow the senescence to proceed until the detached leaf becomes yellow. This yellowing phase is known to be critical for the final properties of the leaf. This phase was analyzed for chemical and expression changes in leaves of cultivars belonging to the three main tobacco types, Virginia, Burley, and Oriental, grown according to their respective practices. In line with important metabolic changes, we observed a significant transition in the genetic program, including chlorophyll degradation, amino acids, and reducing sugar production within hours following the field harvest. We found that in Burley and Oriental tobacco varieties, certain amino acids, such as asparagine, are rapidly synthesized upon detachment, whereas the Virginia tobacco variety mainly produces reducing sugars. Genes differentially expressed after two days of leaf senescence were identified. Downregulated genes mostly belong to metabolic pathways, such as oxidation reduction, carbohydrate and lipid metabolism, and photosynthesis (e.g., RuBisCO subunits). On the other hand, upregulated genes are related to redox reactions, transcription regulation, proteolysis, hydrolase activity, and other catabolic processes. This finding is consistent with previous studies depicting leaf senescence processes. Go-terms analysis performed after 48 hours allowed identification of key transcripts involved in sugar and amino acid metabolism pathways, which may explain some major observed biochemical changes linked to the redistribution of carbon and nitrogen resources during tobacco leaf senescence.