Cytogenetically unrelated clones have been detected by chromosome banding analysis in many breast carcinomas. Because these karyotypic studies were performed on short-term cultured samples, it may be argued that in vitro selection occurred or that small clones may have arisen during culturing. To address this issue, we analyzed 37 breast carcinomas by G-banding and comparative genomic hybridization (CGH), a fluorescent in situ hybridization--based screening technique that does not require culturing or tumor metaphases. All but two of the 37 karyotypically abnormal cases presented copy number changes by CGH. The picture of genomic alterations revealed by the two techniques overlapped only partly. Sometimes the CGH analysis revealed genomic imbalances that belonged to cell populations not picked up by the cytogenetic analysis and in other cases, especially when the karyotypes had many markers and chromosomes with additional material of unknown origin, CGH gave a more reliable overall picture of the copy number gains and losses. However, besides sometimes revealing cell populations with balanced chromosome aberrations or unbalanced changes that nevertheless remained undetected by CGH, G-banding analysis was essential to understand how the genomic imbalances arose in the many cases in which both techniques detected the same clonal abnormalities. Furthermore, because CGH pictures only imbalances present in a significant proportion of the test sample, the very detection by this technique of imbalances belonging to apparently small, cytogenetically unrelated clones of cells proves that these clones must have been present in vivo. This constitutes compelling evidence that the cytogenetic polyclonality observed after short-term culturing of breast carcinomas is not an artifact.