Eighty-seven strains belonging to 18 species of Fusarium were characterized by restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA). The polymerase chain reaction (PCR) was used to amplify a fragment including internal transcribed spacers and variable domains of the 28S rDNA. Data from RFLP analysis of amplified rDNA using eight frequent-cutting restriction enzymes permitted the definition of 23 rDNA haplotypes. The combination of only four restriction enzymes was sufficient to resolve the 23 haplotypes. Each haplotype could be assigned to a single Fusarium species, except for two haplotypes which were common to two or three closely related species. Polymorphism was found within some Fusarium species. Grouping among Fusarium strains derived from restriction analysis was, on the whole, in agreement with other molecular and morphological classification criteria. Therefore, the PCR/RFLP method described in this paper provides a simple and rapid procedure for the differentiation of Fusarium strains at the species level.