In order to improve the exploration and utilization efficiency of wild tobacco germplasm resource, the genetic diversity of 30 wild tobacco accessions was accessed by phenotypic variations and SSR molecular markers. The results revealed abundant phenotypic variations at each of traits, with the genetic diversity index of 0.831 to 1.955. Among them, the diversity index of node distance was the highest, followed by leaf length and width. The marker analysis using 30 pairs of SSR primers detected 98 alleles with an average of 6.125. The mean value of Shannon information index (I), expected heterozygosity (He), observed heterozygosity (Ho) and Polymorphic information content (PIC) were 1.5425, 0.7596, 0.2404 and 0.8668, respectively. The cluster analysis and population structure analysis roughly suggested four groups, likely coincidence with the geographical collection sites. Sixteen SSR markers were used to construct the fingerprints of 30 wild tobacco accessions, which laid a theoretical foundation for the research on the identification system of wild tobacco accessions. Moreover, tests for disease resistance suggested seven accessions including N.exigua were resistant to black shank disease. The species N. nesophila were highly resistance to bacterial wilt, while N. debneyi and N.stocktonii were resistant. Five accessions including N. alata were immune to TMV. Nine accessions including N.alata were resistant to CMV, and 15 accessions including N. debneyi were resistant to PVY. This study established foundation for the identification and evaluation, germplasm resources innovation and explored excellent gene of wild tobacco resources.