Abstract: In order to improve the utilization efficiency and to develop new utilization approaches of air/sun-cured tobacco germplasm resources, we conducted agronomic trait characterization, chemical composition determination and genetic diversity analyses using 93 air/sun-cured tobacco germplasms grown in five tobacco cultivation areas in China. The results showed that the agronomic traits and chemical components of different germplasms varied greatly. The coefficient of variation ranged from 7.60% to 70.17% in different traits. The coefficient of variation of the growth period is the smallest, and that of the total chlorine is the largest. The nicotine content ranged from 1.20% to 8.63%, and potassium content ranged from 0.91% to 4.18%. In addition, 43 pairs of primers with specific amplification were screened from 580 pairs of SSR primers, and 146 polymorphic bands were obtained, of which the polymorphic ratio was 97.99%. The Shannon's index was 0.9379, Nei's diversity index was 0.5356. The 93 tobacco germplasms were clustered into 3 groups with the genetic similarity coefficient of 0.50 by cluster analysis. Germplasms from different cultivation areas in many cases were not clustered together. The genetic consistency between the population in the southwest cultivation area and the Huanghuai cultivation area was 0.9580, while the genetic consistency between the southwest cultivation area and the northeast cultivation area was 0.7820. These results will provide reference for screening and matching the parental germplasms for breeding of excellent mixed cigarette and oral tobacco.