Pathogenicity and genetic variation were investigated within a collection of 104 banana-infecting isolates of Cylindrocladium (teleomorph Calonectria) originating from different countries and representing previously described morphological taxa or species. These root-rot fungi, along with endoparasitic nematodes, have been reported to be causal agents of necrotic lesions that induce root breakage and toppling of banana plants. Little is known about the individual pathogenic effects of the species involved or their genetic diversity. In the present study, among the five morphological taxa found in the banana rhizosphere, only isolates showing an atypical morphology relative to Cylindrocladium gracile (named Cy. gracile-like isolates) and Cy. spathiphylli isolates were pathogenic on banana cultivar 'Grande Naine'. When comparing the latter isolates with others of the same species, but originating from different hosts, an analysis of rDNA spacer polymorphism partitioned isolates of Cy. spathiphylli by host into a banana - tea group and a Heliconia - Spathiphyllum group. Furthermore, isolates from Heliconia were not pathogenic on banana. A pathogenicity assessment of representative isolates from the Cy. gracile-like and the Cy. spathiphylli taxa on six different banana cultivars yielded no evidence of differential interactions between isolates and banana genotypes. Significant differences in susceptibility between banana genotypes were nevertheless detected that could potentially be exploited by breeders. Random amplified polymorphic DNA analysis revealed a genetic similarity ranging from 70% to 100% within Cy. spathiphylli isolates from bananas regardless of the geographic origin. Moreover, Cy. gracile-like isolates were highly similar but showed only 60% similarity relative to the Cy. gracile reference isolates, thus raising questions about their species status.