Filamentous fungi of the genus Cylindrocladium are known since many years to be partly responsible for root necrotic lesions that induce root breakage and toppling disease in banana cropping systems. Until now the implicated taxa have never been typed probably because species are difficult to identify in this genus. Consequently a multiphasic diversity study aimed at characterizing these species and developing a molecular diagnostic tool of Cylindrocladium species was undertaken. Phenotypic and biological analysis of Cylindrocladium isolates from the banana rhizosphere yielded a structuration according to 5 morphotypes with different geographical distribution. Isolates from MTl , MT3, MT4 and MT5 morphotypes could not be clearly identified by their phenotypic traits because of their similarity with different complexes of morphologically similar species. MT2 isolates seemed to be conspecific with the species Cy. spathiphylli. Analysis of ribosomal DNA spacers polymorphism pointed out the conserved nature of ITS region in the genus Cylindrocladium whereas the IGS region displayed polymorphism that can easily be used for discrimination of species. CAPS on the amplified intergenic spacer represent a rapid and reliable molecular diagnostic tool of Cylindrocladium species that can easily be transferred in banana producing zones. It revealed that MT2 and MT5 isolates are respectively conspecific with the species Cy. spathiphylli and Cy. gracile while MT3 and MT4 are related to the species Cy. scoparium and Cy. floridanum sensu lato. Despite their atypical phenotype, MTl isolates were shown to be closely related to the species Cy. gracile. RAPD markers revealed that they only have 60% genetic similarity with this species. Taking into account their overall characteristics, they were further recognized as a related undescribed species called Cy. macrogracile. Pathogenicity evaluation of these 5 taxa towards banana yielded that Cy. spathiphylli and Cy. macrogracile as respecti