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Summary: The study of chromosomes without or with molecular DNA probes provides crucial insight for understanding research findings, as well as refining diagnosis, prognosis, and therapeutics in clinical settings. Here, we present a protocol for chromosome preparation, conventional G-banding, locus-specific fluorescent in situ hybridization, and spectral karyotyping for both mouse and human samples. This protocol optimizes the preparation of chromosomes from mouse and human cells for subsequent conventional and molecular cytogenetic analysis.For complete details on the use and execution of this protocol, please refer to Binz et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.