Although circulating tumour DNA (ctDNA)-based next-generation sequencing (NGS) is a less invasive method for assessingESR1mutations that are essential mechanisms of endocrine therapy resistance in patients with oestrogen receptor-positive breast cancer, adequate amounts of DNA are required to assess polyclonalESR1mutations. By combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay, we have developed a novel detection system to screen for polyclonalESR1mutations in ctDNA. A validation assay was prospectively performed on clinical samples and compared with the NGS results. The PNA-LNA PCR clamp assay was validated using six and four blood samples in whichESR1mutations were detected by NGS and no mutations were detected, respectively. The PNA-LNA assay results were comparable with those of NGS. We prospectively assessed the concordance between the PNA-LNA PCR clamp method and NGS. Using the PNA-LNA PCR clamp method,ESR1mutations were detected in 5 out of 18 samples, including those in which mutations were not detected by NGS due to small amounts of ctDNA. The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for polyclonalESR1mutation detection in the ctDNA of patients with breast cancer.