Tobacco (Nicotiana tabacum L.) is an important agricultural crop plant that plays a significant role in the economies of many countries. Tobacco breeding aims to develop varieties with wide adaptability, higher potential and suitable chemical constituents for the cigarette industry. To explore the genetic potential and select suitable parents it is necessary to assess genetic diversity of tobacco germplasm resources. The aim of this study was to investigate the genetic variability in the CRA-CAT germplasm collection of Nicotiana genus, as revealed by SSR molecular markers. 70 accessions of N. tabacum, including unrelated and related lines and 10 wild Nicotiana species, were examined. Tobacco genotypes were analysed with 35 SSR primer pairs that produced from 1 to 7 bands each with sizes ranging from 70 to 350 bp. A total of 112 bands were generated of which 45 were polymorphic. Among the tobaccos and the wild Nicotiana species, the 35 SSR primer pairs produced from 2 to 16 bands each with sizes ranging from 70 to 350 bp. A total of 237 bands were generated of which 125 were polymorphic. The analysis carried out by UPGMA clustering, based on Jaccard genetic distances, revealed a high rate of genetic similarity among the analysed tobacco genotypes. Genetic variability revealed by SSR analysis at inter and intra-specific level will be discussed.