Forty-four unidentified isolates of Cylindrocladium were recovered from banana root lesions and soils from intensive cropping systems in the Caribbean region, Costa Rica and Cameroon. They were then examined and compared with reference isolates using combinations of morphological characters, sexual crosses and polymorphism of amplified rDNA spacers. According to conidium and vesicle morphology, they were consistently classed into two distinct morphotypes, MT1 and MT2. Sequences of PCR products of their internal transcribed spacer (ITS) region compared with similar sequences from reference isolates representing 7 species of Cylindrocladium revealed a very low polymorphism. By contrast, polymorphism in the amplified intergenic spacer region (IGS), as revealed by RFLP analysis, was found to be consistent with the current taxonomy of Cylindrocladium species, and sufficient to distinguish taxa at the inter- and intraspecific level. Using this IGS typing procedure, MT1 field isolates were found to group unequivocally with Cy. gracile, while MT2 isolates grouped with Cy. spathiphylli. The existence of intraspecific variation in the latter species was clearly demonstrated. Reports of these two species from the banana rhizosphere were confirmed by sexual crosses for Cy. spathiphylli, but not for Cy. gracile, which presently has no known teleomorph. IGS-RFLP markers therefore proved to constitute a rapid and suitable complement to morphological and mating studies for delineation of Cylindrocladium species.