We have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 originated from the MDA-MB-435 breast tumor cell line but have opposite metastatic capabilities in vivo. To investigate gross genetic changes present in this model, we performed a detailed molecular cytogenetic evaluation of the parental cell line, the M4A4 and NM2C5 cell lines, and related clones of metastatic phenotype. Using a combination of spectral karyotyping (SKY), G-banding, and fluorescence in situ hybridization (FISH), we were able to resolve the identity of all common marker chromosomes present in MDA-MB-435 cells, and to define several chromosomal changes, which were specific to each monoclonal cell line. Twenty identical structural and numerical chromosomal aberrations, including trisomies of chromosomes 2 and 5 as well as t(1;7), t(1;10), t(8;11), t(8;15), and t(20;21), were present in all cell lines. The majority of translocations were relatively simple non-reciprocal rearrangements, most frequently involving chromosomes 19, 1, 6, and 20. Chromosomal gains of 1, 7q, 8q, and 20q are common alterations in breast cancer. The metastatic M4A4 cell line contained numerous unique chromosomal aberrations, of which an abnormal banding region on chromosome 22, abr(22), was the best clone-specific identifier. Conversely, the t(12;15)(q22;q26.1) was found exclusively in the non-metastatic NM2C5 cell line. The integration of these karyotypic data with other cytogenetic and genomic databases will enhance our ability to identify genes that play critical roles in cancer development and progression.