Purified DNA from isolates of Fusarium poae, Fusarium sporotrichioides, Fusarium kyushuense and Fusarium langsethiae was used as a template to amplify a 658-bp fragment from the trichodiene synthase (tri5) gene of these fungi with the gene-specific PCR primer pair Tox5-1/Tox5-2. Fragments obtained were isolated and sequenced. DNA sequence alignments revealed high similarity between the sequences derived from F. sporotrichioides and F. langsethiae (98.7%) and less similarity between the latter species and F. poae (90.9%). Phylogenetic analysis of the aligned sequences using the tri5 sequence of Fusarium pseudograminearum as an outgroup revealed clear separation between one group consisting of F. poae and F. kyushuense and another consisting of F. sporotrichioides and F. langsethiae. The two latter species could not be distinguished phylogenetically on the basis of their tri5 sequences. Taxon-specific reverse primers were designed from the aligned sequences and combined with the tri5 gene-specific forward primer Tox5-1. The new reverse primers enabled specific amplification of a fragment of approximately 400 bp from DNA isolated from F. sporotrichioides, F. poae, F. langsethiae and F. kyushuense, respectively. All primers were tested for cross-reactivity with DNA from 26 fungal species potentially capable of producing trichothecenes. Only the primer designed for F. langsethiae cross-reacted with F. sporotrichioides. PCR assays were applied in analysis of artificially and naturally infected samples of barley and oats. On artificially infected barley, species were selectively detected by the corresponding primers. In naturally infected oats, F. langsethiae was identified by the combination of two PCR assays designed for detection of F. sporotrichioides and F. langsethiae, respectively.