Chromosome preparation and fluorescent in situ hybridization (FISH) are indispensable methodologies in cytogenetics, serving as a vital link between molecular biology and genome visualization. In the era of next-generation sequencing and near-complete genome assemblies, these techniques remain crucial for elucidating chromosomal architecture, dynamics, and evolutionary relationships. This chapter presents robust protocols for preparing mitotic and meiotic chromosomes from root tips and floral tissues, optimized for diverse plant species. Detailed procedures for enzymatic digestion, chromosome squashing and spreading, and FISH are described, ensuring reproducible and high-quality results. Furthermore, protocols for probe labeling and fluorescence detection highlight the adaptability of FISH for various cytogenetic applications, including chromosome painting or genomic in situ hybridization. By mastering these approaches, researchers can explore chromosomal organization with unprecedented resolution. These step-by-step protocols are designed to be accessible to both beginners and experienced researchers, ensuring wide applicability across plant species and cytogenetic contexts.