Amplified fragment length polymorphism (AFLP) analysis was used to determine the degree of intra- and inter-specific genetic variation in the genus Nicotiana. Forty-six lines of cultivated tobacco (Nicotiana tabacum L.) and seven wild Nicotiana species, including N. sylvestris, N. tomentosiformis, N. otophora, N. glutinosa, N. suaveolens, N. rustica, and N. longiflora, were analyzed, using at least eight different oligonucleotide primer combinations capable of detecting a minimum of 50 polymorphic bands per primer pair. The amount of genetic polymorphism present among cultivated tobacco lines (N. tabacum) was limited, as evidenced by the high degree of similarity in the AFLP profiles of cultivars collected worldwide. Six major clusters were found within cultivated tobacco that were primarily based upon geographic origin and manufacturing quality traits. A greater amount of genetic polymorphism exists among wild species of Nicotiana than among cultivated forms. Pairwise comparisons of the AFLP profiles of wild and cultivated Nicotiana species show that polymorphic bands present in N. tabacum can be found in at least one of three proposed wild progenitor species (i.e., N. sylvestris, N. tomentosiformis, and N. otophora). This observation provides additional support for these species contributing to the origin of N. tabacum.