The advancement of fluorescence in situ hybridization-based assays has permitted more refined delineation of chromosomal loci involved in complex chromosomal rearrangements (CCRs) and gene amplification. In this detailed molecular cytogenetic analysis, spectral karyotyping (SKY), multicolor banding (mBAND) analysis, and microarray comparative genomic hybridization (CGH) were used to refine the analysis of chromosomes with amplifications and small intrachromosomal rearrangements such as inverted duplications and interstitial deletions present in the osteosarcoma cell line MG-63. SKY analysis has limited resolving power to delineate cryptic chromosomal rearrangements, so mBAND assays were performed for a subset of chromosomes (i.e., 6, 8, 17, and 20). Of the 10 clonal CCRs analyzed in detail with mBAND, 5 were found to have rearrangements between 8q24 and either 6p23 approximately pter or 6p21, with multiple copies of this translocation inserted at various sites in the different chromosomes. In two CCRs, 6p21 and 8q24 generated an alternating pattern of mBAND probe hybridization, indicating the presence of a large coamplified repeat unit within homogeneously staining regions. Microarray CGH analysis demonstrated focal high-level amplification of 8q23 approximately q24, 6p22 approximately pter, and 6p21, in agreement with the pattern of chromosome subband gains identified with mBAND. Thus, sequential SKY, mBAND, and microarray CGH provided a comprehensive description of some of the intricate chromosomal aberrations present in the complex MG-63 karyotype and permitted reconstruction of the fine structure of the genomic rearrangements, thus providing some important mechanistic clues concerning the details of the amplification process in tumors.