AFLP(Amplified Fragment Length Polymorphism) analysis was conducted to cultivars of tobacco, Nicotiana tabacum in order to select the cultivar-specific markers. AFLP results using 12 primer sets revealed genetic diversity among 12 field grown tobacco cultivars. Polymorphic fragments amplified by PCR was purified and cloned to identify their nucleotide sequences. From the sequences of them, 40 primer sets were designed to select cultivar-specific markers. When genomic DNA isolated from tobacco were used as PCR template, a set of primers, BrSF/BrSR showed Burley-specific band patterns. The results indicate that AFLP technique used in this experiments is useful for identifying tobacco cultivars in a rapid manner.