Background:Many breast cancer tumor suppressor genes have been reported to undergo hypermethylation, including ER, RASSF1A, HIC, CDH1, GSPT1, APC, TWIST and CCND2. We first determined the promoter methylation of the above eight tumor suppressor genes as epigenetic markers of breast cancer in three cell lines with different ER status.Methods: This study was an experimental study performed on three cell lines: ER-positive MCF-7, ER-negative MDA-MB-468 and the ER- and HER2-positive breast cancer cell line (BT-474). For this purpose DNA was extracted from these cell lines and gene promoter methylation was analyzed by methylation-specific PCR.Results: Our results showed that all studied genes were hypermethylated in ER- negative MDA-MB-468 cells. We detected hypermethylation of GSPT1, RASSF1A and CCND2 genes in these three cell lines. In addition, the ER-positive MCF-7 cells showed methylation of TWIST and APC genes.Conclusion: The numbers of methylated molecular biomarkers is increased with tumor progression.This finding may be important in breast cancer therapy for different ER status.