The development of fluorescence in situ hybridization (FISH) has led to the advancement of chromosome studies not only for physical mapping and genome analyses but also as a tool for evolutionary studies. Isolated repetitive DNA sequences have been useful cytological markers, but large-insert genomic libraries (e.g., bacterial artificial chromosomes BACs, yeast artificial chromosomes YACs, and cosmids) are being increasingly used to serve as probes for large segments of DNA across related genomes. Although FISH is usually applied to metaphase chromosomes, fiber FISH, a variation of FISH using extended DNA fibers, is now used to measure loci at the resolution of a few kilobases to compare orthologous genome segments across related genomes. A generalized set of protocols for chromosome preparation, FISH, and fiber FISH are presented; however, it is often necessary to experiment with techniques for different plant taxa for successful molecular cytogenetic studies.