Genetic variation of host specificity among isolates of Fusarium oxysporum f. sp. vasinfectum is identified with an elaborate set of differential host species and cultivars that distinguish six races of the fungus. These differentials include four plant species besides three species of cotton (Gossypium), and many of the cultivars are difficult or impossible to obtain. Thus, it is impossible to identify races with the original cultivars developed for this purpose. Complementation among mutants for utilization of nitrate is an alternate method to identify form species and variation within form species of F. oxysporum. This technique was used to determine variation in isolates of F. oxysporum from Upland cotton (Gossypium hirsutum L.) in the USA. Isolates were obtained from culture collections, from plants (with and without wilt symptoms) grown on cotton field soils from NC, SC, GA, FL, AL, MS, AK, LA, and TX, and from Bauerman funnel extracts of the same soils. Totals of 300 and 1200 isolates of F. oxysporum from plants and soils, respectively, were tested for virulence to 'Rowden' and 'Stoneville 825' cultivars using stem puncture inoculations of plants with 4 to 6 true leaves. Seventy-two isolates from plants and none of those from soil caused wilt disease characterized by extensive vascular discoloration. All of the 300 isolates from plants were tested for genetic compatibility using complementary nit-1 and nit-M mutants. The ATCC type specimens of race 1 and 2 (from SC) were not compatible with each other or with any of the other 70 isolates that caused cotton wilt. The 70 isolates were assigned to 3 vegetative compatibility (V-C) groups with distinct A, B, and C subgroups in one group. Thus, at least five different V-C groups cause Fusarium wilt of Upland cotton in the USA. We have designated the major group, which has three subgroups, as V-C 1 with the next most prevalent groups designated as V-C 2 and 3; race I and 2 type specimens apparently are indigenous only to parts of South Carolina and are designated as V-C 4 and 5, respectively. None of the 228 avirulent isolates or type specimens of races 3, 4, or 6 (from foreign countries) were compatible with the five V-C that caused wilt of cotton in the USA. The V-C 1 group occurred throughout the Cotton Belt and apparently is responsible for most of the Fusarium wilt in the USA- The V-C 1, 2, and 3 groups displayed similar patterns of virulence to 'Rowden', 'Stoneville 825,' 'Acala 44,' and 'Acala Royale' cultivars of G. hirsutum and were more virulent than the race 1 and race 2 type specimens. None of the V-C groups were virulent to Gossypium barbadense 'Seabrook Sea Island 12B2.'