We report the characterization, by reverse fluorescence in situ hybridization (FISH), of 59 hybrids retaining fragments of human chromosome 5. Most of these hybrids are radiation hybrids generated by gamma irradiating, at low dosage, a monochromosomal hybrid retaining chromosome 5 as its only human contribution. The partial chromosome paints generated from these hybrids will make powerful tools for cytogenetic investigations, especially on the cytogenetic evolution of primates, and examples are reported. The molecular characterization of these hybrids was refined using 74 sequence-tagged sites (STSs), which allowed the physical dissection of chromosome 5 into 71 distinct regions with an average length of 2.7 Mb. The panel, therefore, is also suitable for high-precision subregional mapping of new genes or sequences located on chromosome 5. As an additional resource for cytogenetic studies involving chromosome 5, we report the characterization, by FISH, of 73 YACs from CEPH. The vast majority of these YACs are recognized by at least one of the STSs used for hybrid characterization, thus enabling the integrated use of YACs and partial chromosome paints derived from the hybrids.