Triple negative breast cancer (TNBC) is an aggressive subtype with limitations in therapy. Although cyclin dependent kinase inhibitors (CDKi) have been proven in breast cancer, challenges remain in TNBC. Successful inhibition of CDK4/6 relies on intact Rb tumor suppressor (encoded by tumor suppressor gene RB1). However, in addition to gene mutation or deletion, RB1, as an imprinted gene, also has a mechanism of inactivation due to loss of imprinting (LOI). This study aimed to ascertain the imprinting status of RB1 in TNBC.We applied bioinformatic analyses to evaluate methylation differences on the RB1 imprinting control region CpG85 among subtypes of breast cancer. Deregulation of RB1 expression by LOI in TNBC cell lines was further tested by RT-qPCR with stimulation by 5-aza-2-deoxycytidine (DAC) treatment. In addition, RB1 CpG85 methylation levels of circulating cell-free DNA (cfDNA) in plasma was assessed by pyrosequencing in 15 enrolled TNBC patients and 6 non-cancer donors. Survival analysis based on TCGA and GEO databases were performed to explored the potential clinical significance.Bioinformatic analysis showed hypomethylation at CpG85 of RB1 in TNBC, which was further confirmed in cell lines. LOI of RB1 affected its transcription. Analysis of cfDNA showed CpG85 was differentially methylated in TNBC patients, with 6/15 patients displaying hypomethylation at cg18481241 and 1/15 at cg03085377 within CpG85. Patients with hypomethylation of these sites correlated with worse overall survival.RB1 exhibits potential LOI in TNBCs, laying the groundwork for more precise subtyping and treatment of TNBC patients.© 2025. The Author(s).