Cylindrocladium leaf blight is one of the most important diseases affecting Eucalyptus grandis plantations in Colombia. Disease symptoms include both leaf blotch and shoot blight and these can lead to severe defoliation. This reduces the productivity of E. grandis in forestry zones with high humidity. The objective of this study was to identify the Cylindrocladium spp. associated with Cylindrocladium leaf blight of E. grandis in three important forestry regions of Colombia. Isolates were obtained from samples collected within these areas and morphology as well as DNA sequence data were used for identification. Results of both morphological comparisons and analysis of β-tubulin gene sequences showed that only C. spathulatum was associated with the disease symptoms in the evaluated areas. Evaluation of a Eucalyptus clonal trial showed that clones differ greatly in their susceptibility to infection by C. spathulatum. This presents excellent opportunities for disease avoidance in future.