SummaryThe morphological similarity, relatively small size of the chromosomes and low metaphasic indexes obtained in root meristems have hindered karyotypic characterization and the application of banding techniques in Coffea. We have developed a method based on the use of cell suspension aggregates treated with amiprophos-methyl (APM) and macerated in enzymatic solution. This method generated cytogenetic preparations in which the chromosomes showed well-defined primary and secondary constrictions, facilitating the pairing of homologues and assembly of the karyogram, as well as the identification of active NOR and heterochromatin associated with the secondary constriction. This alternative technique could help on the analysis of other species with similar karyotypic characterization problems.