Open AccessTobacco (Nicotina tabacum) has been introduced to Thailand for hundreds of years. All tobaccos cultivated in the country are legally separated to local (or early-imported) and imported cultivar groups. However, no method could precisely differentiate the two groups, especially from cured leaf samples. Amplified fragment length polymorphism (AFLP) analysis was introduced to estimate genetic polymorphism of 19 tobacco cultivars grown in Thailand. Thirty-two selective primer-combinations were screened on the genomic DNA extracted from cured leaves. Three primer pairs were selected and resulted in 139 scorable AFLP fragments, of which 103 (74.1%) were polymorphic. Genetic relationship analysis revealed clustering patterns of tobacco samples generally following the cultivar groups. Almost all local cultivars were found closely related to Burley and Turkish types of the imported group, but significantly separated from Virginia type. Our finding therefore should be an important knowledge for further research on cultivar identification and genetic improvement of tobaccos.