Cell division is a highly regulated process. Checkpoints can halt cell‐cycle progression due to adverse conditions such as misalignment of chromosomes to prevent missegregation. The search for new regulators of the cell cycle revealed the mitotic checkpoint gene CHFR (checkpoint with forkhead‐associated and ring finger). CHFR coordinates an early mitotic phase by delaying chromosome condensation in response to a mitotic stress. Because aneuploidy and chromosome instability are common in malignant breast tumors, we screened 24 breast cancer cell lines for CHFR expression and demonstrated that 50% (12 of 24) of breast cancer cell lines had low CHFR levels. Expression of CHFR was reactivated with the demethylating agent 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) in two low‐CHFR–expressing cell lines. Eleven of these 12 (92%) low‐CHFR–expressing cell lines had an unusually high number of condensed chromosomes and high mitotic indices in response to nocodazole treatment. Transfection of CHFR in one of these cancer cell lines lowered the mitotic index after nocodazole treatment. In conclusion, our data suggested that low CHFR expression associated with high mitotic indices in response to nocodazole treatment were common in the breast cancer cell lines studied. Additional flow cytometry studies and analysis of a protein that interacts with CHFR in vitro, polo‐like kinase 1 (PLK1), suggests that this CHFR‐associated early G₂/M checkpoint is complex, involving additional, as yet unidentified, proteins. Further analysis of CHFR in breast cancer cells will be important for understanding the complex mechanisms leading to aneuploidy and chromosomal instability observed in breast cancer. © 2003 Wiley‐Liss, Inc.