Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy-related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G-banding. To locate the chromosomal breakpoints, DAPI-counterstained band images from all metaphases were transformed to G-band-like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G-banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G-banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of -5/5q-and -7/7q-) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with -5/5q-and in 4 of 8 with -7/7q-, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G-banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS.