The hemibiotrophic fungus Colletotrichum kahawae is the causal agent of Coffee Berry Disease - CBD, an economically devastating disease restricted to Arabica coffee production in Africa. Understanding pathogen variability is crucial for the implementation of disease control measures. In this study, six isoenzymatic systems (esterase, acid and alkaline phosphatase, malate dehydrogenase, peroxidase and superoxide dismutase) were used to assess the genetic variation among 12 C. kahawae isolates (from different geographic origins and with different levels of aggressiveness towards coffee) and one Colletotrichum gloeosporioides isolate (not pathogenic to green berries). Cluster analysis of the banding profiles obtained for the six enzymes enabled the differentiation of the two species and revealed the existence of intraspecific variability among C. kahawae isolates. Alkaline phosphatase was the most discriminative enzyme, allowing also the discrimination between the most and least aggressive isolates of C. kahawae.