Unidentified strains of Cylindrocladium that were baited from soil in the Amazonas state of Brazil or isolated from Adiantum in the Netherlands were examined morphologically and analysed phylogenetically in comparison with reference strains. Phylogenetic trees inferred from the 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA, as well as the beta-tubulin gene, separated species in accordance with their morphological features and characteristics. Although species differences based on ITS sequences were consistent, there were only a few informative sites available, making it difficult to clearly identify the unknown strains. Better resolution in separation of species was achieved from the beta-tubulin data. The strains baited from soil in Brazil were found to represent two species, namely Cylindrocladium gracile (Bugnic.) Boesew. and Cylindrocladium pseudogracile Crous. Morphologically, these two species are similar, except that the latter has slightly narrower, 1(-3)-septate conidia, and produces a Calonectria teleomorph. Furthermore, the strain from the Netherlands represents Cylindrocladium rumohrae El-Gholl & Alfenas, a species previously known only from Panama. Results of this study also indicated that the beta-tubulin gene is phylogenetically more informative than the ITS regions for distinguishing species of Cylindrocladium.