Amplified fragment length polymorphism (AFLP) was conducted on a set of 92 Nicotiana tabacum L. accessions from diverse types (flue-cured, dark air-cured, burley, oriental, and cigar wrapper) and breeding origins to identify markers associated with disease resistances. Eleven primer combinations were required to identify 33 polymorphic fragments. This allowed the identification of 92% of these accessions, and yielded sufficient information for building a neighbor joining tree. Clusters of accessions with common traits or breeding origins were observed. An important part of this polymorphism could be related to interspecific introgressions from other Nicotiana species, performed during the breeding history of N. tabacum to confer resistance to pathogens. Seven fragments were associated with three different resistances: two for the blue-mold ( Peronospora tabacina Adam) resistance derived from Nicotiana debneyi Domin, two for the Va gene (Potato Virus Y susceptibility), and three for the black root rot ( Chalara elegans ) resistance of N. debneyi o rigin. Some of these markers were converted into sequence characterized amplified region markers, and validated on recombinant inbred lines or doubled-haploid lines.