Hypermethylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, the present study focused on the DNA methylation profile of invasive breast carcinoma. The aim of the study was to evaluate the prognostic significance of Ras association domain family 1 isoform A (RASSF1A) promoter methylation status in operable breast cancer, and to analyze the utility of this biomarker regarding its association with metastatic and nonmetastatic axillary nodal status. For this purpose, formalin-fixed, paraffin-embedded tissue specimens from 116 breast cancer patients with known axillary nodal status were subjected to assessment of RASSF1A promoter methylation status by methylation-specific polymerase chain reaction (MSP) and methylation-sensitive high-resolution melting assay, and the results were subsequently validated by bisulfite sequencing. A multinomial logistic regression model was used to model the dependence of distinct levels of methylation status of the RASSF1A promoter on the nodal status. Promoter region CpG hypermethylation was identified by MSP in 97 (83.6%) of 116 primary breast tumors, while hypermethylation of RASSF1A was confirmed by MS-HRM in 107 (92.2%) of 116 cases of breast cancer. Based on the results of the multinomial logistic regression model, there was no significant difference between the frequency of RASSF1A promoter methylation and axillary lymph node status of patients in general. However, upon adjustment of pN stage, an association was identified between pN0 lymph node-negative status (without axillary metastases) and percentage of RASSF1A methylation in two groups of heterogeneous methylated alleles with ≤50% methylated (P<0.05) and >50% methylated alleles (P<0.0001). If a patients' nodal status changes from pN- to pN+ then the risk of having >50% methylated alleles increases by 7%. The present study revealed a specific phenomenon, suggesting that the presence of heterogeneous methylated alleles in the RASSF1A gene is significantly associated with lymph node-negative status in breast cancer patients. Furthermore, greater significance with negative axillary nodal status was observed with a higher level of heterogeneous methylated alleles in the RASSF1A gene.