In order to catalog and preserve 63 tobacco germplasm accessions, which were collected by Chongqing Project Group in a frame of the Third National Survey and Collection Action on Crop Germplasm Resources, we conducted the field experiments for identifying the phenotypic and marker-assisted genetic diversity. Morphological characteristics and main agronomic traits were surveyed and analyzed. These tested germplasm accessions represented abundant genetic diversity at both quantitative traits and qualitative traits. Specially for qualitative traits, leaf shape had the highest diversity index, followed by Leaf length, leaf width and stem girth. Clustering analysis suggested four groups of these newly-collected tobacco germplasm accessions. The germplasms resided to the first group were mainly rustica, while the germplasms from the third group showed higher plant height and bigger leaves with a potential for high-yield breeding. Tests for disease resistance indicated that 11 accessions were resistant to TMV and CMV. Particularly, two of them were free of TMV infection and highly resistant to CMV, which could be used for disease resistance breeding. By taking use of field phenotypic datasets and genotyping results using SSR markers, the germplasm accessions showing identical were excluded for cataloglization and conservation into the National Bank of Tobacco Germplasm Resources. Therefore, survey and collection of tobacco germplasm resources is of great significance, in order to enrich the genetic diversity of Tobacco Germplasm Resources and benefit for tobacco breeding.