Fusarium wilt (FW) is one of the most economically damaging cotton diseases worldwide, causing yellowing, wilting, defoliation, vascular tissue damage and ultimately death. Identification of molecular markers linked to FW genes is vital to incorporate resistance into elite cotton cultivars. An intraspecific F(2) in Gossypium hirsutum L. was developed by crossing with a highly resistant cultivar Zhongmiansuo 35 (ZMS35) and a susceptible cultivar Junmian 1 to screen simple sequence repeats (SSRs) closely linked to the FW resistance gene. FW was identified in F(2:3) families by evaluating seedling leaf symptoms and vascular tissue damage at plant maturity under natural field infection conditions over 2 years. The results showed that FW resistance segregated in a 3:1 ratio as a simple monogenic trait in F(2:3) families. Molecular mapping identified a FW resistance gene closely linked with the SSR marker JESPR304(-280) in chromosome D3(c17). We proposed to name this gene FW ( R ). A composite interval mapping method detected four QTLs for FW resistance in Chr.A7(c7), D1(c15), D9(c23) and D3, respectively. Among them, one major QTL (LOD > 20) was tagged near marker JESPR304 within an interval of 0.06-0.2 cM, and explained over 52.5-60.9% of the total phenotypic variance. The data confirmed the existence of a major gene in Chr.D3. This is the first report of molecular mapping of a major gene contributing FW resistance in cotton. The present research therefore provides an opportunity to understand the genetic control of resistance to FW and conduct molecular marker-assisted selection breeding to develop FW resistant cultivars.