For investigating the differences in leaf microstructure among flue-cured tobacco cultivars, the microstructures of mature middle leaves of cv. K326, Honghuadajinyuan (HD), Yunyan87 (Y87), Yunyan85 (Y85), G80, Zhongyan14 (Z14) and Xiangyan1 (X1) cultured under the same conditions were analyzed in detail. The results showed that the leaves of Y85 and X1 were the thickest, and those of HD, Y87, Z14 were the thinnest. Z14 had the largest ratio of palisade to spongy tissue and larger clearance, however the differences among the cultivars were not obvious. While the morphology of stoma differed significantly among cultivars, K326 were moderate both in leaf structure and stoma morphology. It was speculated that the gene type and ecological adaptation were the main factors causing the differences of leaf structure among cultivars.