We developed single copy probes from the draft genome sequence for fluorescence in situ hybridization (scFISH) which precisely delineate chromosome abnormalities at a resolution equivalent to genomic Southern analysis. This study illustrates how scFISH probes detect cryptic and subtle abnormalities and localize the sites of chromosome rearrangements. scFISH probes are substantially shorter than conventional recombinant DNA-derived probes, and Cot1 DNA is not required to suppress repetitive sequence hybridization. In this study, 74 single copy sequence probes (>1,500 bp) have been developed from ≥100 kb genomic intervals associated with either constitutional or acquired disorders. Applications of these probes include detection of congenital microdeletion syndromes on chromosomes 1, 4, 7, 15, 17, 22 and submicroscopic deletions involving the imprinting center on chromosome 15q11.2q13. We demonstrate how hybridization with multiple combinations of probes derived from the Smith-Magenis syndrome interval on chromosome 17 identified a patient with an atypical, proximal deletion breakpoint. A similar multi-probe hybridization strategy has also been used to delineate the translocation breakpoint region on chromosome 9 in chronic myelogenous leukemia. Probes have also been designed to hybridize to multiple cis paralogs, both enhancing the chromosomal target size and detecting chromosome rearrangements, for example, by splitting and separating a family of related sequences flanking an inversion breakpoint on chromosome 16 in acute myelogenous leukemia. These novel strategies for rapid and precise characterization of cytogenetic abnormalities are feasible because of the sequence-defined properties and dense euchromatic organization of single copy probes. © 2003 Wiley-Liss, Inc.