Cancer genes are often deregulated by genomic rearrangements. Accordingly, analysis of the participant chromosomes responsible now occupies a key role in characterizing and identifying cancer cell lines. Cytogenetics may also be used to study the nature and extent of chromosome breakage induced by radiation or chemicals (“clastogenesis”), to distinguish individual cells or clones within a tumor cell population and to monitor the stability of chromosome rearrangements. This chapter describes cytogenetic procedures for characterizing cancer cells in culture. Cell lines allow the use of a wider range of harvesting and hypotonic treatments to optimize metaphase chromosome preparations than that possible with primary cultures. This assists improved banding, fluorescence in situ hybridization (FISH), and Spectral Karyotyping (SKY) analysis for research, rendering cell lines ideal tools for oncogenomics, ideally in parallel with transcriptomic analysis of the same cells. The experience of the writers with more than 800 cell lines has shown that no single hypotonic harvesting protocol is adequate consistently to deliver satisfactory chromosome preparations. Thus, evidence-based protocols are described for hypotonic harvesting, rapid G-banding, and FISH and SKY analysis of cell cultures to allow troubleshooting and fine-tuning to suit the requirements of individual cell lines.