Abstract: To examine the differences in the type, secretion composition and genetic regulation mechanisms of leaf trichomes between Nicotiana sylvestris and Nicotiana tomentosiformis, the leaf trichomes were observed and counted by electron microscopy, the composition of glandular trichome secretions were analyzed by gas chromatography-mass spectrometry (GC-MS), and trichome development and the expression levels of glandular trichome secretions synthesis related genes were analyzed by real-time fluorescent quantitative PCR (qRT-PCR). The results showed that the leaf surface of N. tomentosiformis was mainly covered by protective trichomes and short stalk glandular trichomes, and long stalk glandular trichomes were rare. The leaf surface of N. sylvestris was mainly covered by long glandular trichomes. The content of cembranoid diterpenoids in the glandular trichome secretions of N. sylvestris was significantly higher than that of N. tomentosiformis, and sucrose esters were not detected in the glandular trichome secretions of the latter. The key genes associated with the synthesis of cembranoid diterpenoids, labdanoid diterpenoids and sucrose esters were highly expressed in N. sylvestris. The trichome development related genes NtARF3, NtCycB2, NtAquaporin, NtMIXTA and NtBCR2a were highly expressed in N. tomentosiformis, while NtHD1 and NtSAP1 were highly expressed in N. sylvestris. In summary, during the evolution of N. sylvestris and N. tomentosiformis, the trichome phenotypes were differentiated and different metabolic adaptative strategies were formed. The differences were determined by the expression patterns of key genes associated with the synthesis of glandular trichome secretion and the regulatory network of trichome genetic development.