ABSTRACT Wheat head blight caused by Gibberella zeae (anamorph: Fusarium graminearum) is a threat to food safety in China because of mycotoxin contamination of the harvested grain, the frequent occurrence of the disease, and the failure of chemical control in some areas due to benzimidazole resistance in the pathogen population. The molecular resistance mechanism, however, of G. zeae to benzimidazole fungicides (especially carbendazim; active ingredient: methyl benzimidazol-2-yl carbamate [MBC]) is poorly understood. DNA sequences of a beta-tubulin gene (beta(2)tub) (GenBank access number FG06611.1) in G. zeae were analyzed. Mutations in beta(2)tub in moderately resistant strains (MBC(MR)) included TTT (Phe)-->TAT (Tyr) at codon 167 or TTC (Phe)-->TAC (Tyr) at codon 200. A highly resistant strain (MBC(HR)) had two point mutations, one at codon 73, CAG (Gln)-->CGG (Arg), and the other at codon 198, GAG (Glu)-->CTG (Leu). To confirm that mutations in the beta(2)tub confer resistance to benzimidazole fungicides, the entire beta(2)tub locus was deleted from MBC(MR) and MBC(HR) strains of G. zeae. The resulting Deltabeta(2)tub mutants from both MBC(MR) and MBC(HR) strains grew normally on MBC-free potato dextrose agar medium and were supersensitive to MBC. Complementation of the Deltabeta(2)tub mutants by transformation with a copy of the intact beta(2)tub locus from their parent strains exhibited less resistance than the original strains, and complementation of the Deltabeta(2)tub mutants by transformation with a copy of the intact beta(2)tub locus from sensitive strains restored MBC sensitivity. The results indicated that the mutations in the beta(2)tub gene conferred resistance of G. zeae to benzimidazole fungicides and this gene can be used as a genetic marker in G. zeae.