Abstract: To explore the phenotypic differences and molecular regulatory mechanisms in the upper leaves of flue-cured tobacco cultivars Cuibi 1, K326, and Yunyan 87 under the same fertilization level, we combined agronomic trait determination, conventional chemical component analysis, transcriptome sequencing, and qRT-PCR validation to investigate the phenotypic variations and transcriptional response characteristics of upper leaves among these three cultivars. The results showed that the leaf area, dry weight, total nitrogen, total alkaloid, potassium, and protein contents in upper leaves of Cuibi 1 were significantly higher than those of K326 and Yunyan 87; Yunyan 87 exhibited the highest accumulation of total sugar and reducing sugar, while K326 had a relatively higher starch content. KEGG pathway enrichment analysis indicated that differentially expressed genes (DEGs) among cultivars were significantly enriched in pathways related to plant hormone signal transduction, substance metabolism, mineral absorption, and secondary metabolism. GO functional enrichment analysis revealed that DEGs had specific enrichment at the cellular component and molecular function levels, with all covering nitrogen, phosphorus and potassium transport and metabolism-related functions. Combined with the validation of core gene expression levels in nitrogen, phosphorus and potassium metabolism, highly expressed members of nitrogen utilization-related genes (NtNRT1, NtAMT1.3, NtLHT1) and potassium transporter genes (NtHAK1, NtKC1) were identified in Cuibi 1; nicotine synthesis genes (NtQPT, NtODC) were relatively active in K326; the overall expression levels of core genes in nitrogen, phosphorus and potassium metabolic pathways were lower in Yunyan 87. In conclusion, significant differences exist in upper leaf phenotypes, chemical component accumulation and transcriptional responses among Cuibi 1, K326 and Yunyan 87 under the same fertilization condition, and the differential expression of nitrogen, phosphorus and potassium metabolism-related genes may be the key factor leading to the inter-cultivar differences.