Background: Normal cell division is coordinated by a bipolar mitotic spindle, ensuring symmetrical segregation of chromosomes.Cancer cells may occasionally divide into three or more directions, known as multipolar mitosis (MM).Such phenomenon has been proposed to generate genetic diversity and thereby contribute to clonal evolution in tumor cells.This assumption has, however, not been validated experimentally.Principal Findings: Chromosome segregation and DNA content in daughter cells from MM were assessed by multiphoton cross sectioning and fluorescence in situ hybridization in cancer cells and non-neoplastic transformed cells.The DNA distribution resulting from multipolar cell division was found to be highly variable, with frequent nullisomies in the daughter cells.Time-lapse imaging of H2B/GFP-labelled multipolar mitoses revealed that the time from the initiation of metaphase to the beginning of anaphase was prolonged and that the metaphase plates often switched polarity several times before metaphase-anaphase transition.Apart from this,several genes essential for mitotic spindle assembly checkpoint were assessed by immunofluorescence during mitotic process in both normal bipolar cells and MM cells.It was shown that metaphase-anaphase transition in MM cells was accompanied by a normal reduction of cellular cyclin B levels, but typically occurred before completion of the chromatid separation, which is in a sharp contrast to normal bipolar cells where reduction of CCNB following completion of chromatid separation.Furthermore the observation that Centromeric AURKB and MAD2 foci frequently remained on the centromeres of multipolar ana-telophase chromosomes indicated that multipolar mitoses have capacity to by pass the spindle assembly checkpoint with some sister chromatids remaining unseparated after anaphase.This was further supported by the finding that a high frequency of nondisjunction events, resulting in a near-binomial allotment of sister ehromatids to the daughter cells was found while scoring the distribution of individual chromosomes in multipolar daughter nuclei.Conclusion: The capability of multipolar mitoses to circumvent the spindle assembly checkpoint system typically results in a near-random distribution of chromosomes to daughter cells.Spindle multipolarity could thus be a highly efficient generator of genetically diverse minority clones in transformed cell populations.