Knowledge of the genetic background in tobacco cultivars is very important not only for genetic evaluation in breeding but also for management of germplasms and protection of cultivars. DNA marker technology is one of the powerful tools for such purposes. Simple Sequence Repeat (SSR) markers were developed for identification and discrimination of tobacco cultivars. It is known that SSR markers are abundant in plant genomes, and variations of the repeat number among different lines within a species occur at high frequency. These characteristics, along with an evenly dispersed genomic distribution, are properties which make SSR markers ideal genetic markers. The SSR-enriched genomic libraries for AG-, CT- and TAA-repeat motifs were constructed using the magnetic beads method based on the hybridization between the biotin-labeled probes and the target nucleic acid molecules. Following determination of the DNA sequences of clones in each library, primer pairs specific for SSR loci were synthesized. After the primers were tested for the presence of polymorphism among eight tobacco cultivars, the resulting functional and polymorphic SSR markers were tested for 84 tobacco cultivars consisting of flue-cured, Burley, Oriental and other types.26 out of 36 SSR markers were identified for use in discriminating the 84 cultivars. To facilitate the genotyping of cultivars, a multiplex PCR system was employed in which the 26 SSR markers were sorted and bulked into 5 groups. These SSR markers were confirmed to be useful the genotyping of DNA prepared not only from fresh materials but also from cured leaves and cut laminas in commercial cigarettes.