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Study on the genetic bio-diversity of tobacco 39 cultures including Burley tobacco 16 cultivars 15 and 8 local and wild cultivars respectively, was conductive at Maejo University. 20 markers were employed for identification the genetic diversity. The result showed that 13 SSR primers could presented the polymorphic band for 60 alleles, which are average of 3 alleles per SSR primers. The SSR markers showed the difference between allele clearly, They were NBS133, NBS141, NBS148, NBS155 NBS162, NBS173, NBS191, NBS209, NBS226, NBS244, NBS267, NBS271 and NBS329. And grouping the relationship of 39 cultivars tobacco by NTSY spec version 2.02, following the clustering by SAHN and dendogram by UPGMA (unweighted pair group method with arithmetic) from the above tobacco could be classified into 3 groups: group 1 was commercial cultivars, group 2 local cultivars and group 3 wild tobacco cultivars. For the furthered tobacco breading programs, we can selected the difference of genetic base of parent line (cultivars) to bred the new tobacco cultivate for high yield, quality and insect disease resistance cultivars.