Nonrandom chromosomal aberrations, particularly in cancer, identify pathogenic biological pathways and, in some cases, have clinical relevance as diagnostic or prognostic markers. Fluorescence and colorimetric in situ hybridization methods facilitate identification of numerical and structural chromosome abnormalities. We report the development of robust, unique-sequence in situ hybridization probes that have several novel features: 1) they are constructed from multimegabase contigs of yeast artificial chromosome (YAC) clones; 2) they are in the form of adapter-ligated, short-fragment, DNA libraries that may be amplified by polymerase chain reaction; and 3) they have had repetitive sequences (eg, Alu and LINE elements) quantitatively removed by subtractive hybridization. These subtracted probes are labeled conveniently, and the fluorescence or colorimetric detection signals are extremely bright. Moreover, they constitute a stable resource that may be amplified through at least four rounds of polymerase chain reaction without diminishing signal intensity. We demonstrate applications of subtracted probes for the MYC and EWS oncogene regions, including 1) characterization of a novel EWS-region translocation in Ewing's sarcoma, 2) identification of chromosomal translocations in paraffin sections, and 3) identification of chromosomal translocations by conventional bright-field microscopy.