This chapter focuses on the whole-chromosome hybridization, and covers a subset of in situ hybridization methods in which the aim is to stain a chromosome or chromosomes by using a collection of nucleic acid fragments specific for the chromosome(s) in question. Whole-chromosome hybridization is a subgroup of in situ hybridization methods in which the aim is to stain specifically complete chromosomes. In situ hybridization allows a given nucleic acid sequence to be seen in its normal cellular, nuclear, or chromosomal context. This technique is a specialized form of nucleic acid hybridization and as such involves the selection of nucleic acid probes, labeling of probes, hybridization to the specimen, removal of nonspecifically bound probe, and finally, the detection and visualization of the hybridizing probe. The ability to perform whole-chromosome hybridization experiments depends entirely upon the availability of nucleic acid probes which bind to specific chromosomes. These can take the form of genomic DNA, used, for example, to identify human chromosomes in human-rodent hybrid cell lines.