Centromere-specific multi-color FISH (cenM-FISH) is a new multicolor FISH technique that allows the simultaneous characterization of all human centromeres by using labeled centromeric satellite DNA as probes. This approach allows the rapid identification of all human centromeres by their individual pseudo-coloring in one single step and is therefore a powerful tool in molecular cytogenetics. CenM-FISH fills a gap in multicolor karyotyping using WCP probes and distinguishes all centromeric regions apart from the evolutionary highly conserved regions on the chromosomes 13 and 21. The usefulness of the cenM-FISH technique for the characterization of small supernumerary marker chromosomes with no (or nearly no) euchromatin and restricted amounts of available sample material is demonstrated in prenatal, postnatal, and tumor cytogenetic cases. In addition, rarely described markers with the involvement of heterochromatic material inserted into homogeneously staining regions could be identified and characterized by using the cenM-FISH technique.