Fusarium wilt caused by soil-borne fungus Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is an early season disease causing root rot, and seedling wilt and death. However, it was unknown if cotton (Gossypium spp.) growth stages and root wounding affected genotypic responses to FOV4. The objectives of this study were to investigate the effects of cotton growth stage, cultivar, and root wounding on FOV4associated disease development at 7-, 14-, 21-, and 28-d post inoculation through five replicated tests in controlled environmental conditions with temperatures at 21 to 23 ˚C. Seedlings inoculated at the cotyledon stage incurred the highest disease incidence (DI), disease severity rating (DSR), and mortality (MR), followed by seedlings inoculated at the 1-, 3-, and 5-true leaf stages in descending order. Root wounding at the cotyledon stage followed by immediate inoculation with FOV4 inoculum made all the tested cultivars and lines highly susceptible. Among the six cultivars tested, FM 2334GLT and Pima DP 359 RF were the most resistant, Pima S-7 was highly susceptible, and Acala 1517-08, Acala 1517-18 GLS, and Pima PHY 881 RF were susceptible. The importance of artificial inoculation at a true leaf stage with or without root wounding to identify resistance that may be undetected under field conditions was discussed.