Purpose: Multicolor FISH (M-FISH) was introduced in 1996 to scan all 24 chromosomes in different fluorescent colors by use of a specific filter set and computer software. However, the clinical utility of M-FISH has been limited because of the lack of commercial availability of reagents and hardware. We have evaluated M-FISH for identification of markers, derivative chromosomes, and complex karyotypes.Methods: We present our findings based on a representative sample of one normal and six abnormal cases from a variety of tissue types. The results of M-FISH were confirmed by other well-established FISH probes.Results: M-FISH analyses were successful in all six cases. The derivative chromosomes, ring, and a complex karyotype were resolved.Conclusions: We find M-FISH to be an invaluable tool for a high degree of accuracy and efficiency for chromosome identification. The limitations similar to spectral karyotyping system (SKY) include the inability to detect intrachromosomal anomalies, abnormalities involving the p-arms of acrocentrics and areas rich in highly repetitive DNA. In addition, there are some concerns of misinterpretation due to overlap of fluorophore combinations of different chromosomes, especially for subtle insertional translocations.