The identities of Botryosphaeriaceae causing cankers on Eucalyptus in Colombia were investigated using morphological and DNA sequence comparisons. The pathogenicity of the species was also assessed on 42 Eucalyptus grandis clones planted at four different sites. Two species of the Botryosphaeriaceae were found to occur on E. grandis in Colombia. Neofusicoccum ribis was the more common species, and also the most pathogenic. Botryosphaeria dothidea was found only in one zone of Colombia, and was also less pathogenic than N. ribis. These two species could be distinguished easily based on DNA sequences of the ITS1/ITS2 rDNA region and EF1-α, in addition to conidial sizes. Significant differences in resistance of clones to these pathogens were also evident from the various trials.