Chromosomal aberrations are common in cancers. However, the search for chromosomal aberrations leading to development of specific solid tumors has been severely hindered because the majority of solid tumors have complex chromosomal aberrations that differ within the same tumor types. A similar phenomenon exists in immortalized cell lines. The underlying mechanisms driving these diverse aberrations are largely unknown. Telomeres play crucial roles in protecting the integrity of eucaryotic chromosomes and maintaining genomic stability of human cells. Telomere lengths on individual chromosomes in normal human somatic cells are heterogeneous and undergo progressive shortening with aging process. In this study, for the first time, a molecular cytogenetic method using sequential telomere quantitative fluorescence in situ hybridization and spectral karyotyping on the same human metaphases was applied successfully to examine the dynamic profiles of individual telomere shortening and their relationship to chromosome aberrations in multiple human cell lines undergoing immortalization. Human ovarian surface epithelial cells and esophageal epithelial cells were immortalized by the expression of HPV16 E6 and E7, which drive cells to proliferate by inactivating p53 and Rb genes. In these cell lines, we consistently detected large-scale differences in telomere signal intensities not only among nonhomologous chromosome arms but also between some homologous chromosome arms. The cell lines derived from different donors had different profiles of critically short telomeres (lacking telomere signals). Strikingly, the different profiles of chromosomal structural aberrations in multiple immortalized cell lines were highly significantly associated with the distinct distributions of critically short telomeres in whole-genome. Since cellular immortalization is one of the hallmarks of cancer, our findings suggest that distinct profiles of critically short telomeres in different human individuals might play an essential role in determining the complex and individual-specific chromosomal structural aberrations in human solid tumors.