Cytogenetic characterization of murine chromosomes using banding techniques like R- or G-banding is technically demanding due to the similar size and the acrocentric structure of all chromosomes. The molecular cytogenetic technique of spectral karyotyping (SKY) overcomes that difficulty by karyotyping metaphase chromosomes after different and simultaneous fluorescence labeling of the whole genome. SKY allows the detection and identification of numerical as well as structural chromosome aberrations with a resolution of approximately 2 Mb. The technique is applicable to all fast-proliferating cells, e.g., cells of the hematopoietic system like stem cells or T- and B-lymphocytes. It is also applicable to murine embryonic fibroblast or cells isolated from tissues with increased proliferation-especially tumor tissues. Furthermore, SKY is recommended for the cytogenetic characterization of newly established cell lines.