The genus Fusarium includes several species that produce trichothecenes. We analyzed DNA sequences from a variable region at the 5' end of the large nuclear ribosomal DNA (rDNA) (28S) to determine the genetic relatedness of trichothecene-producing Fusarium species. All trichothecene-producing strains clustered together, and two monophyletic groups were resolved. The first clade included strains of F. acuminatum, F. sambucinum, F. tumidum, F. compactum, F. camptoceras (red pigment), F. sporotrichioides, and F. venenatum, which produced type A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol, and diacetoxyscirpenol). The second clade consisted of strains of F. crookwellense, F. culmorum, and F. graminearum, which produced type B trichothecenes (fusarenone-X, nivalenol, and deoxynivalenol). The phylogenetic placement of the species based on rDNA correlated better with toxic secondary metabolite data rather than with the current classification system based on morphology.