Fusarium wilt of cotton, caused by the soilborne fungus Fusarium oxysporum f. sp. vasinfectum (FOV),is a widespread and devastating disease occurring in most cotton-growing regions of the world. The most successful strategy to manage Fusarium wilt is the use of resistant cultivars. Recently, two sources of germplasm, MD25-26ne and MD25-27, which have superior yield, fiber quality, and resistance against Fusarium wilt and root-knot nematodes (Meloidogyne spp.), were released. The purpose of this study was to evaluate the response of eight Upland cotton (including MD25-26ne and MD25-27) and two Pima cultivars (susceptible and resistant controls) against seven genotypes of FOV (races 1, 2, 3, 4, and 8 and genotypes LA108 and LA140) in a greenhouse trial. To inoculate plants, roots of 2-wk-old seedlings of each cultivar were dipped for 4 min in a conidial suspension (106 conidia/mL) of each FOV genotype. Based on dry shoot weights and extent of vascular discoloration; Phytogen 800 followed by MD25-26ne and MD25-27 were the most resistant cultivars against multiple genotypes of FOV. FOV race 4 caused mild symptoms on PHY 800, PHY 98M-2983, MD25-26ne, and MD25-27 but caused severe symptoms on DP 744 and Rowden. In addition, Rowden was highly susceptible to FOV races 1, 2, and 8 and LA108 and LA140. Phytogen elite lines PHY x1, PHY x2, and PHY x3 were moderately susceptible to multiple genotypes of FOV. These data indicate that PHY 800, MD25-26ne, and MD25-27 can be recommended for planting in fields with previous histories of Fusarium wilt and can be used in breeding programs as sources of resistance to Fusarium wilt.