Abstract: We cloned two solanesyl diphosphate synthase genes, NtSPS1 and NtSPS2, from tobacco (Nicotiana tabacum cv. Honghuadajinyuan) plants to investigate their role in tobacco solanesol biosynthesis. In this study, we performed sequence alignment, phylogenetic analysis, and subcellular localization analysis of NtSPS1 and NtSPS2. Further, we studied the expression levels of NtSPSs in different organs of tobacco plants, and analysed the relationship between solanesol, chlorophyll content and the expression levels of NtSPSs in different organs of tobacco plants. The results showed that:NtSPS1 contained an open reading frame (ORF) of 1209 bp encoding 402 amino acids and NtSPS2 contained an ORF of 1206 bp encoding 401 amino acids. Both NtSPS1 and NtSPS2 had two conserved DDxxD domains, which were related to the function of NtSPS. The homology among NtSPS1, NtSPS2, and SlSPS was very high, probably because both tobacco and tomato were solanaceous crops. Both NtSPS1 and NtSPS2 were located in the chloroplast. The expression levels of NtSPS1 and NtSPS2 in different organs of tobacco plants decreased according to this order:leaf > stem > root, which was consistent with the distribution of solanesol and chlorophyll in tobacco plants. This study could lay a foundation for further research on the role of NtSPSs in the regulation of tobacco solanesol biosynthesis.