Chromosomal instability, characterized by abnormal numbers or structures of chromosomes, is a common feature of human cancers, but the mechanisms behind these changes are still unclear. Since centrosomes play a pivotal role in balanced chromosomal segregation during mitosis, we attempted to investigate the association between centrosome abnormalities and chromosomal instability in a large number of human pancreatic cancer cell lines. Immunofluorescence microscopy revealed centrosomes that were highly atypical with respect to their size, shape, and number in most cell lines. These abnormal centrosomes contributed to the assembly of multipolar spindles, resulting in defective mitosis and chromosome mis-segregation. Interestingly, a high frequency of centrosome defects inversely correlated with the growth rate of cells in culture. Fluorescence in situ hybridization revealed a dramatic variation of chromosome numbers in cell lines with the defective centrosome phenotype. Furthermore, a significant positive correlation existed between the level of centrosome defects and the level of chromosomal imbalances. These results indicate that centrosome abnormalities can lead to spindle disorganization and chromosome segregation errors, which may drive the accumulation of chromosomal alterations. Thus, defects in centrosome function may be an underlying cause of genetic instability in human pancreatic cancers.