Beginning with the hypothesis by von Hansemann and Boveri that cancer is a disease of the chromosomes (1), cytogenetic analysis was applied to cancer cells, including mouse models of human cancer, yet failed for more than 50 years to provide conclusive evidence for a causative effect of chromosomal aberrations in tumorigenesis. This was first due to technical limitations, then to insurmountable preconceptions, which even prevented the enumeration of the correct number of human chromosomes until Tijo and Levan's report in 1956 (2). The establishment of this baseline invigorated cytogenetic research, and shortly thereafter the association of specific chromosomal aneuploidies with disease syndromes, such as Down syndrome, Edward syndrome, and Pätau syndrome was demonstrated (3–5). A defining moment in cancer cytogenetics was the description of a non-random, specific aberration in patients with chronic myelogenous leukemia – the so-called Philadelphia chromosome – by Nowell and Hungerford (6). This aberration was later shown by Janet Rowley (7) to be a balanced translocation between chromosomes 9 and 22 and established the paradigm of translocation-induced activation of oncogenes. One invaluable technical leap in chromosome analysis was achieved by the introduction of chromosome banding by Caspersson and Zech in 1969, the adaptation to human chromosomes, and the introduction of Giemsa banding (8–10). These achievements made possible not only the ability to correctly enumerate chromosomes but also to assess their structural integrity. The value of this discovery cannot be over-estimated, and catalogs of chromosomal aberrations in leukemia, lymphoma, and solid tumors are convincing proof of the importance of this advancement. The recently published third edition of the compendium Cancer Cytogenetics by Heim and Mitelman reports that some 50 000 cases have been studied employing chromosome banding techniques, and that banding analysis has made it possible to identify many fusion genes at the site of chromosomal translocations, including such notorious oncogenes as MYC in Burkitt's lymphoma (11). However, with the development of molecular biological methods such as DNA cloning and hybridization – the first in situ hybridization was reported by Gall and Pardue in 1969 (12) – the discipline of molecular cytogenetics emerged, lending unprecedented flexibility to experimental design and dramatically improving resolution. Four of the most relevant molecular cytogenetic techniques for the analysis of cancer chromosomes and genomes, namely, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY) and M-FISH, comparative genomic hybridization (CGH), and interphase cytogenetics, will be reviewed here in detail.