Species of Calonectria represent an important group of plant pathogenic fungi that cause serious losses to crops in tropical and subtropical climates. Because these fungi are difficult to identify, selected species were subjected to DNA analysis with the aim of producing a more objective identification technique. This was done by amplifying the 5′ end of the β-tubulin gene from isolates representing more than 30 species of Calonectria. A neighbour joining analysis was performed on a total data set of 86 isolates, while a representative subset of isolates was analysed by means of maximum parsimony. Both analyses yielded dendrograms with concordant topology. Several clades were strongly supported by bootstrap, confirming the morphological and biological species concepts presently employed. At least two large groupings were evident and both contained small-spored, 1-septate species. The only morphological character that correlated with DNA phylogeny was vesicle shape. These data support Calonectria as a monophyletic genus with Cylindrocladium anamorphs. Furthermore, this study also emphasises the importance of vesicle morphology in identifying Cylindrocladium anamorphs, and hence species of Calonectria.