Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of ‘Delgold’ tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a ‘Delgold’ background, and ‘PB19’, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between ‘Delgold’ and ‘PB19’, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between ‘Delgold’ and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between ‘Delgold’ and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.