Background: Various studies have analyzed the relationships between new biological parameters and DNA ploidy for human cancer. However, the biological significance of DNA ploidy remains unclear, since there have so far been few studies analyzing DNA ploidy in relation to the combination of multiple such biological parameters. Methods: Samples for the analysis of DNA ploidy, estrogen receptor (ER), progesterone receptor (PgR), p53 protein and epidermal growth factor receptor (EGFR) were prepared from the same frozen specimens of 498 primary breast cancers. DNA ploidy was determined by flow cytometry, while ER and PgR were determined by an enzyme immunoassay. Both p53 protein and EGFR were determined by immunohistochemical analyses. Results: Aneuploidy was significantly correlated with the absence of ER, the absence of PgR, the positivity of p53 protein and the positivity of EGFR, and it was also significantly correlated with the combination of the absence of ER and PgR and the combination of the positivity of p53 protein and EGFR. A deviation in the four parameters was defined as the absence of ER, the absence of PgR, the positivity of p53 protein or the positivity of EGFR. As the number of deviations of these four biological parameters increased, the incidence of aneuploidy increased significantly (p < 0.0001). Multivariate analysis also indicated that deviation of two, three or all four parameters were significant factors for DNA ploidy, while the p value decreased and the odds ratio increased as the number of deviations of the four biological parameters increased. Conclusions: DNA aneuploidy based on flow cytometric analysis reflects the accumulation of deviations in the four biological parameters investigated in the present study. The two facts that DNA aneuploidy reflects the accumulation of the aggressiveness of these biological parameters and that DNA aneuploidy consisted of heterogeneous characteristics which were represented by the deviation of each biological parameter should be taken into consideration when selecting treatment strategies for breast cancer.