Fluorescence in situ hybridization (FISH) has become an increasingly important method for assessing chromosome rearrangement. The reciprocal translocation constituting the Philadelphia (Ph) chromosome [t(9;22)(q34;q11)] characterizes more than 90% of patients with chronic myelogenous leukemia (CML). However, in the remaining cases the Ph chromosome (genetically characterized by the fusion of the BCR-ABL genes) is thought to arise through complex translocations that are often not readily apparent using routine chromosome-banding analysis. For this reason we have developed unique band-specific probes for two-color FISH that detect unequivocally the Ph chromosome, and its derivatives. Results of the application of these probes are illustrated by analysis of 11 cases of CML (9 of which contain "variant" translocations). The probes were generated by chromosome microdissection and in vitro amplification of the bands involved in the Ph translocation, leading to an extremely fast and sensitive approach to identify this alteration in leukemic cell populations.