Colletotrichum kahawae is the causal agent of Coffee Berry Disease (CBD), which is capable of infecting green berries on Arabica coffee. Although this disease is currently restricted to Africa, it represents a threat to cultivation in America and Asia. The thorough understanding of Colletotrichum biology will certainly contribute to better suited disease management approaches. Potential differences in genomic composition and karyological analysis may contribute to deepen the knowledge on the infection process, such as specific pathogenicity factors in extra chromosomes, and to contribute to the taxonomic placement of the CBD pathogens in the C. kahawae cluster within the 'gloeosporioides' species complex. In this work, a combination of karyological and genomic tools were used to further characterize C. kahawae. Several isolates representing the genetic and geographical diversity of the species were analyzed using different approaches that include chromosome separation and characterization by pulse field gel electrophoresis, cytogenetic analysis and chromosome number estimation by microscopy and genome size determination by flow cytometry. Differences among C. kahawae isolates were identified concerning the genome size, which ranged 74-89 Mb, and the number of minichromosomes (below 2 Mb), from 1 to 5.