Abstract: To study the nicotine accumulation and mature characteristics of low-nicotine tobacco and discern the correlation between nicotine content and the tobacco's mature traits, NC95 and K326 and their low-nicotine tobacco varieties (MAFC5, MLAFC34, LAFC53 in NC95 and CD01 in K326 background) were utilized as experimental materials. Effects of varying nitrogen application levels on the nicotine content of tobacco leaves across different stages and leaf positions were analyzed. The findings revealed a gradual increase in nicotine content in low-nicotine tobacco as the plants matured. Notably, the nicotine content of low-nicotine tobacco varieties was lower compared to the control sets NC95 and K326, with MAFC5 > MLAFC34 > LAFC53 > CD01, exhibiting reductions of 39%, 43%, 79% and 96% respectively, compared to the controls. Additionally, the nicotine content of leaves of MAFC5, MLAFC34 and LAFC53 increased from bottom to top, while in CD01, it decreased from bottom to top. Furthermore, the nicotine content in the leaves of MAFC5, MLAFC34 and LAFC53 increased of 24.3%、26.1% and 19.6% from low nitrogen application to high nitrogen application respectively, whereas CD01 displayed only 9.9% increase. Regarding maturity characteristics, MLAFC34 and LAFC53 displayed poorer traits compared to CD01 and MAFC5, which were closer to those of the control tobacco. Our study underscores that the nicotine content in the leaves of low-nicotine tobacco varieties is lower than that of control varieties. Moreover, the effects of nitrogen application on the nicotine content varied among all varieties, indicating no inherent correlation between nicotine content and mature characteristics.