<jats:title>Abstract</jats:title><jats:p>The range of secondary metabolites (SMs) produced by the rice pathogen <jats:italic>Fusarium fujikuroi</jats:italic> is quite broad. Several polyketides, nonribosomal peptides and terpenes have been identified. However, no products of dimethylallyltryptophan synthases (DMATSs) have been elucidated, although two putative <jats:italic>DMATS</jats:italic> genes are present in the <jats:italic>F. fujikuroi</jats:italic> genome. In this study, the in vivo product derived from one of the DMATSs (DMATS1, FFUJ_09179) was identified with the help of the software MZmine 2. Detailed structure elucidation showed that this metabolite is a reversely N‐prenylated tryptophan with a rare form of prenylation. Further identified products probably resulted from side reactions of DMATS1. The genes adjacent to <jats:italic>DMATS1</jats:italic> were analyzed; this showed no influence on the biosynthesis of the product.