To direct the effort to improve the sucrose ester composition in the secretions of glandular trichomes of tobacco cultivar K326, the high-secreting tobacco (Nicotiana tabacum) germplasm T.I.1068 was used as donor parent, while the flue-cured tobacco cultivar K326 was used as recurrent parent. Multiple generations of backcrossing and self-pollination were performed, with selection in each generation assisted by molecular markers linked to the Ⅲ-Ⅴ type sucrose ester synthase gene (BMVSE). Combined with observations of agronomic traits and analysis of leaf surface chemical components, a near-isogenic line of K326 with increased sucrose ester types, named SEK326, was developed. The agronomic traits of SEK326 differed significantly from those of T.I.1068, but were basically similar to those of K326, except for increased stem to leaf angle and significantly increased internode length. The trichome density of SEK326 was significantly lower than that of T.I.1068, but about 0.39 times higher than that of K326. In addition, the glandular head of SEK326 was fuller and more deeply stained with rhodamine dye. Compared to K326, the contents of cembranoid diterpenes in SEK326 decreased by 18.15%, and the contents of type Ⅰ-Ⅱ sucrose esters decreased by 33.48%. However, with the addition of type Ⅲ-Ⅴ sucrose esters, the total amount of sucrose esters increased by approximately 3.12 times, and there was a significant increase in the content of glandular trichome secretion. qRT-PCR analysis showed that the expression levels of the key gene NtCBTS for cembranoid diterpene synthesis and the key genes NtASAT1 and NtASAT2 for type Ⅰ-Ⅱ sucrose ester synthesis were all decreased compared to K326. Compared to T.I.1068, the total amount of glandular trichome secretion was significantly decreased in SEK326, and Z-abienol and labdene diol were absent from its composition. Molecular detection revealed that both SEK326 and K326 had the same single nucleotide mutation (G 1542 T) in the key gene NtCPS2 for labdene diterpene synthesis, resulting in premature termination of protein translation.