Squamous cell carcinoma of the head and neck and its subset, oral squamous cell carcinoma (OSCC), arise through a multistep process of genetic alterations as a result of exposure to environmental agents, such as tobacco smoke, alcoholic beverages, and viruses, including human papillomavirus. We and others have shown that the karyotypes of OSCC are near-triploid and contain multiple structural and numerical abnormalities. However, despite a background of clonal chromosomal aberrations, individual cells within a culture express many nonclonal numerical and structural abnormalities, termed chromosomal instability (CIN). To evaluate CIN in oral cancer cells, we isolated clones from two OSCC cell lines and carried out classical cytogenetic analysis, fluorescence in situ hybridization using centromere-specific probes, and spectral karyotyping. We observed variation in chromosome number within clones and between clones of the same cell line. Although similar numbers of centromeric signals for a particular chromosome were present, "homologs" of a chromosome varied structurally from cell to cell (marker chromosome evolution) as documented by classical and spectral karyotyping. In addition to the numerical chromosome variations within a clone, we observed marker chromosome evolution by structural chromosome alterations. It appears that both intrinsic structural alterations and extrinsic cytoskeletal factors influence chromosome segregation, resulting in individual tumor cells that express unique karyotypes. We show that CIN and marker chromosome evolution are essential acquired features of neoplastic cells. Proliferation of this heterogeneous cell population may provide some cells with the ability to evade standard therapies.