Abstract: In order to investigate the different utilization of soil nitrogen sources in fertilizer by upper leaves of flue-cured tobacco varieties in tobacco planting areas in southeast Guizhou, the nitrogen accumulation and ammonia volatilization in the upper leaves of cv. Yunyan89 and cv. K326 were measured using ¹⁵NH₄¹⁵NO₃ fertilizer (base fertilizer, 70%) and K¹⁵NO₃ fertilizer (top dressing fertilizer, 30%), both labeled with ¹⁵N isotope. The results indicated that the nitrogen content in the upper leaves of the two varieties increased at first and then decreased during growing, and the proportion of the nitrogen absorbed from soil in the total nitrogen content increased gradually. The contribution of nitrogen absorbed from soil to the nitrogen nutrition needed by the upper leaves was enhanced. The ammonia volatilization from the upper leaves of cv. Yunyan87 was higher than that of cv. K326, and the nitrogen source of ammonia volatilization in the upper leaves of the two varieties was mainly from the nitrogen in fertilizer. It is concluded that the soil nitrogen played a decisive role in the nitrogen nutrition of the upper leaves and the utilization of the upper leaves to the nitrogen in soil and nitrogen in fertilizer differed between the tobacco varieties.