Abstract: To reveal the genetic differentiation of tobacco germplasm among the tested populations, and to screen germplasms with excellent agronomic traits and desirable aroma, 142 tobacco germplasms were selected, and genotyped and analyzed in this study. Results showed that the agronomic and aroma quality traits had a relatively rich diversity, the coefficient of variation for aroma quality traits (23.55%-30.91%) were higher than that for agronomic traits (18.51%-24.34%), and there were significant positive correlations among multiple traits. At a Euclidean distance of 20, 142 germplasms were divided into 5 groups with different characteristics, among which group I had the highest content of chlorogenic acid and rutin, group Ⅲ had the highest plant height and leaf number, group IV had the highest values of middle leaf length, middle leaf width, stem girth and crypto chlorogenic acid content, and group V had the highest content of β-carotene. Six germplasm with excellent comprehensive traits (パィンハルト, Century 1, Beinhart 1000-1, FC8, Turkey M type and Laifengdaniuzi) were selected by combining principal component analysis and membership function analysis. These germplasms exhibited improved balance between agronomic and aroma quality traits, making them valuable for future tobacco breeding programs.