Abstract: To explore the phenotypic differences and molecular regulatory mechanisms of the upper leaves of flue-cured tobacco cultivars Cuibi 1, K326, and Yunyan 87 under the same fertilization level, a combination of agronomic trait determination, routine chemical component analysis, transcriptome sequencing, and qRT-PCR validation was employed to analyze phenotype, chemical composition accumulation, and transcriptional response characteristics of the upper leaves of the three tobacco cultivars. The results showed that the leaf area, dry weight, and contents of total nitrogen, total alkaloids, potassium, and protein of upper leaves of Cuibi 1 were significantly higher than those of K326 and Yunyan 87, and Yunyan 87 exhibited the highest accumulation of total sugar and reducing sugar. KEGG pathway enrichment analysis indicated that differentially expressed genes (DEGs) among the cultivars were significantly enriched in the pathways related to plant hormone signal transduction, substance metabolism, mineral substance absorption, and secondary metabolism. GO functional enrichment analysis revealed that DEGs exhibited species-specific enrichment at the cellular component and molecular functional levels, and were involved in the transport and metabolism of nitrogen, phosphorus, and potassium. qRT-PCR validation further showed that the expression levels of genes related to nitrogen uptake and transport (NtNRT1, NtAMT1.3, NtLHT1) and potassium transport genes (NtHAK1, NtKC1) were significantly upregulated in Cuibi 1. Nicotine synthesis genes (NtQPT, NtODC) were expressed actively in K326. The overall expression levels of genes in metabolic pathways of nitrogen, phosphorus, and potassium were lower in Yunyan 87. In summary, under the same fertilization conditions, there were significant differences in the phenotype, chemical composition accumulation, and transcriptional response of the upper leaves of Cuibi 1, K326, and Yunyan 87. The differential expression of genes related to the metabolism of nitrogen, phosphorus, and potassium may be the key factor leading to inter-cultivar differences.