Abstract: Low sugar and high nicotine contents in tobacco leaves are very important in reducing tar content of cigarettes and improving the strength and aroma of tobacco products. It is also an important raw material of new tobacco products such as e-cigarettes and oral cigarettes. In order to screen high nicotinic flue-cured tobacco germplasm resources, the chemical composition of 30 flue-cured tobacco germplasm resources originally labeled as high nicotinic were reidentified in the national tobacco germplasm Repository. Seven high nicotinic and low sugar germplasm were obtained including Chunleiyihao, Bijinyihao, 8100, I-35, Guangdonghuang (1), Liaoyanshisihao, and NC729. The genetic diversity of 30 high nicotinic germplasm materials was analyzed based on agronomic and chemical traits. The results showed that the coefficient of variation of quantitative traits was 12.79%-44.60% and the genetic diversity index was 1.97-2.35, which showed a high genetic diversity. Based on phenotypic data, principal component analysis, and cluster analysis, the selected germplasm were clustered into five groups. Each group has significant phenotypic traits, with groups Ⅰ, Ⅲ, Ⅳ being classified as low sugar, high nicotine varieties.