Cytogenetic analysis provides critical information of diagnostic and prognostic importance for haematological malignancies. In fact, the identification of recurring chromosomal breakpoints in leukaemias and lymphomas has expedited the cloning of genes whose translocation-induced deregulation causes malignant transformation. The pillar of karyotype analysis rests on chromosome banding techniques that have the distinct advantage that the entire genome can be analysed in a single experiment. However, poorly spread or contracted metaphase chromosomes and highly rearranged karyotypes with numerous marker chromosomes, common in tumour cell preparations, are often difficult to interpret unambiguously and subtle chromosomal aberrations, in particular the exchange of telomeric chromatin or small insertions remain elusive. Fluorescence in situ hybridization (FISH) overcomes some of these limitations, but is mainly utilized to confirm the presence of previously characterized or suspected aberrations. We have developed a novel approach, termed spectral karyotyping or SKY based on the hybridization of 24 fluorescently labelled chromosome painting probes that allows the simultaneous and differential colour display of all human chromosomes. We have used SKY to complement conventional banding techniques in haematological malignancies by analysing 15 cases with unidentified chromosome aberrations. In all instances SKY provided additional cytogenetic information, including the identification of marker chromosomes, the detection of subtle chromosomal translocations and the clarification of complex chromosomal rearrangements. Thus, SKY in combination with standard chromosome banding allows the characterization of chromosomal aberrations in leukaemia with unprecedented accuracy.