<jats:title>Abstract</jats:title><jats:p><jats:italic>Fragments of polyketide synthase (PKS) genes were amplified from complementary DNA (cDNA) of the fusarin C producing filamentous fungi</jats:italic> Fusarium moniliforme <jats:italic>and</jats:italic> Fusarium venenatum <jats:italic>by using degenerate oligonucleotides designed to select for fungal PKS</jats:italic> C<jats:italic>‐methyltransferase (</jats:italic>C<jats:italic>MeT) domains. The PCR products, which were highly homologous to fragments of known fungal PKS</jats:italic> C<jats:italic>MeT domains, were used to probe cDNA and genomic DNA (gDNA) libraries of</jats:italic> F. moniliforme <jats:italic>and</jats:italic> F. venenatum. <jats:italic>A 4.0 kb cDNA clone from</jats:italic> F. venenatum <jats:italic>was isolated and used to prepare a hygromycin‐resistance knockout cassette, which was used to produce a fusarin‐deficient strain of</jats:italic> F. venenatum <jats:italic>(kb=1000 bp). Similarly, a 26 kb genomic fragment, isolated on two overlapping clones from</jats:italic> F. moniliforme<jats:italic>, encoded a complete iterative Type I PKS fused to an unusual nonribosomal peptide synthase module. Once again, targeted gene disruption produced a fusarin‐deficient strain, thereby proving that this synthase is responsible for the first steps of fusarin biosynthesis.</jats:italic>