AbstractChromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and chromosome arms. Chromosome preparation is the basic technique for studies in cytogenetics, including the analysis of chromosomal behavior, chromosomal variation, karyotyping analysis, fluorescence in situ hybridization, immunostaining, and artificial chromosome technology. In this unit, we describe the basic protocol for rice (Oryza sativa) metaphase chromosome preparation from rice root tips, alternative chromosome preparation protocols using cultured cells, and different chromosome spreading techniques. © 2016 by John Wiley & Sons, Inc.