Fluorescence in situ hybridization (FISH) provides an effective means to delineate chromosomes and their subregions during all stages of the cell cycle using fluorochrome-labeled DNA probes on suitable cellular preparations. Probes can be designed to loci of interest, which makes FISH particularly useful for studying whole chromosomes and chromosome subregions/loci in species with minute genomes and/or weak chromosome condensation at metaphase. The latter being a feature of model organisms, such as e.g., the budding yeast Saccharomyces cerevisiae or the fission yeast Schizosaccharomyces pombe. Since its introduction in yeast cytology in 1992, FISH with composite whole chromosome or locus-specific probes in combination with immunofluorescent staining has become a powerful tool in the analysis of chromosome behavior in mitotic and meiotic metaphase and interphase cells. This article outlines a FISH protocol that has proven useful in the study of chromosome behavior of wild-type and mutant yeast strains.