Objective: This study was conducted to determine the relationship between p16 gene methylation and expression of relevant receptors in breast cancer (BC) for subtyping the disease. Methods: Methylation-specific PCR (MSP) was carried out to detect the methylation status of p16 gene in 240 tissue samples and 205 serum samples from BC patients treated at our hospital. Immunohistochemistry was used to determine the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Receiver operating characteristics (ROC) curve was analyzed for diagnostic value based on methylation status for triple-negative (TN) BC. Results: The overall methylation rates of the p16 gene were 36.7% (88/240) and 35.1% (72/205) in the tissue and serum samples, respectively. In patients with ER, PR and HER2-TNBC, the methylation rate of the p16 gene was significantly higher than that in non-triple negative patients (84.9%, 62/73) vs (25.9%, 35/135, P 0.01). The methylation of p16 gene was negatively associated with the expression of ER, PR and HER2 (r = -0.661, -0.694 and -0.765, respectively, P 0.05), but it was not correlated with the pathological characteristics of BC, such as tumor grade and lymph-node metastasis. Receiver operator characteristic (ROC) curve analysis showed that p16 gene methylation had a significant diagnostic value for TNBC with an AUC of 0.815. Therefore, p16 gene methylation is associated with the subtype of TNBC and can be used as an easy and non-invasive approach to screen patients for TNBC.