As a member of the Third National-wide Investigation of Crop Germplasm Resources Project, Hunan team collected 32 tobacco germplasms after systematic investigations. In this study, 23 germplasms were selected from the collected tobacco germplasms, and 22 agronomic traits were identified and analyzed for genetic diversity. The results showed that the tested germplasms had a high level of genetic diversity, and the quantitative traits had a general high diversity index. The three quantitative traits with the highest diversity index were node distance, leaf number, and leaf width. As for qualitative traits, leaf shape had the highest diversity index. Petiole had the highest CV (coefficient variation), followed by plant height and leaf width. Based on the results of cluster analysis, the newly-collected tobacco germplasms were divided into 3 groups. The germplasms from the second group had the most leaves, and the ones from the third group were the tallest, which could be used for breeding varieties with high yield or used as intermediate materials. The identification of the resistance to the major diseases of the characteristic tobacco germplasm demonstrated that 13 germplasms showed resistance to black shank, TMV or CMV, two of which showed pleiotropic resistance to both black shank and TMV, and they could be used for disease resistance breeding. Thus, it is of great significance to carry out the census and collection of tobacco germplasm resources, which can provide richer parental materials or intermediate materials for the breeding of new varieties.