Knowledge of the inheritance of disease resistance and genomic regions housing resistance (R) genes is essential to prevent expanding pathogen threats such as Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans] in cotton (Gossypium spp.). We conducted a comprehensive study combining conventional inheritance, genetic and quantitative trait loci (QTL) mapping, QTL marker-sequence composition, and genome sequencing to examine the distribution, structure and organization of disease R genes to race 1 of FOV in the cotton genome. Molecular markers were applied to F(2) and recombinant inbred line (RIL) interspecific mapping populations from the crosses Pima-S7 (G. barbadense L.) × 'Acala NemX' (G. hirsutum L.) and Upland TM-1 (G. hirsutum) × Pima 3-79 (G. barbadense), respectively. Three greenhouse tests and one field test were used to obtain sequential estimates of severity index (DSI) of leaves, and vascular stem and root staining (VRS). A single resistance gene model was observed for the F(2) population based on inheritance of phenotypes. However, additional inheritance analyses and QTL mapping indicated gene interactions and inheritance from nine cotton chromosomes, with major QTLs detected on five chromosomes [Fov1-C06, Fov1-C08, (Fov1-C11 ( 1 ) and Fov1-C11 ( 2)) , Fov1-C16 and Fov1-C19 loci], explaining 8-31% of the DSI or VRS variation. The Fov1-C16 QTL locus identified in the F(2) and in the RIL populations had a significant role in conferring FOV race 1 resistance in different cotton backgrounds. Identified molecular markers may have important potential for breeding effective FOV race 1 resistance into elite cultivars by marker-assisted selection. Reconciliation between genetic and physical mapping of gene annotations from marker-DNA and new DNA sequences of BAC clones tagged with the resistance-associated QTLs revealed defenses genes induced upon pathogen infection and gene regions rich in disease-response elements, respectively. These offer candidate gene targets for Fusarium wilt resistance response in cotton and other host plants.