Isolates of the type-A trichothecene producing Fusarium sporotrichioides and Fusarium langsethiae were grouped and differentiated in a phylogenetic tree using ITS sequence dissimilarity. An attempt was made to develop a PCR-based assay for the detection and differentiation of Fusarium sporotrichiodes from other Fusarium species using the 5'-region of the tri5 gene as a template. However, this assay was unable to differentiate, to a satisfactory level, between isolates of Fusarium sporotrichioides and Fusarium langsethiae, providing further genetic evidence for their close genetic relationship. A robust and repeatable PCR-assay was developed for the detection and differentiation of both species based on sequence determined from differentially amplified RAPD-PCR products. These assays were able to detect both species in samples of grain taken from the field.