For >3 decades, Giemsa banding of metaphase chromosomes has been the standard karyotypic analysis for pre- and postnatal diagnostic applications. However, marker chromosomes or structural abnormalities are often encountered that cannot be deciphered by G-banding alone. Here we describe the use of multiplex-FISH (M-FISH), which allows the visualization of the 22 human autosomes and the 2 sex chromosomes, in 24 different colors. By M-FISH, the euchromatin in marker chromosomes could be readily identified. In cases of structural abnormalities, M-FISH identified translocations and insertions or demonstrated that the rearranged chromosome did not contain DNA material from another chromosome. In these cases, deleted or duplicated regions were discerned either by chromosome-specific multicolor bar codes or by comparative genomic hybridization. In addition, M-FISH was able to identify cryptic abnormalities in patients with a normal G-karyotype. In summary, M-FISH is a reliable tool for diagnostic applications, and results can be obtained in </=24 h. When M-FISH is combined with G-banding analysis, maximum cytogenetic information is provided.