Background: Circulating tumour cells are a real-time reflection of the ad hoc relevant subpopulation in patients with progressive disease. The study comprises the clinical application of a liquid biopsy to assess the PIK3CA genotype at a single cell level. Methods: Using CellSearch and DEPArray we purified single and groups of CTCs and WBCs from peripheral blood in 29 patients with metastatic hormone receptor-positve breast cancer. Recovered cells were subjected to Ampli1 whole genome amplification (WGA). Temporally-matched circulating cell-free DNA (cfDNA) was purified from plasma. Additionally, gDNA from archival primary tumour (PT) tissue sections was extracted as comparator. Mutation analysis was performed via targeted amplicon sequencing (TAS) of exons 9 and 20. Results: Archival PT tissue section showed a high frequency of mutant PIK3CA (16/27 (59,2%)), with a poor and fair agreement with cfDNA (n = 21; 43% disparity; κ = 0,113) and CTCs (n = 22; 27% disparity; κ = 0,394), respectively. A concordant PIK3CA status between different compartments was observed in 10/18 (56%) samples. At the used sequencing depth, cfDNA failed to the detect PIK3CA mutations in 4 cases (22%), of which three were present in the respective PT and corresponding CTCs. Gain of mutation was observed in 4/18 patients (22%), with a wild type PT and mutant CTCs at progression (cfDNA confirmed the MT genotype in three cases). A wild-type PIK3CA sequence in recovered WBCs indicates a high specificity and tumorigenic nature of the picked up variants. Intra-CTC analysis reveals PIK3CA mutational heterogeneity with the presence of both mutant and wild-type CTCs. Additionally, unique double-mutated CTCs were detected in 10/26 (38%) cases as well. Conclusions: PIK3CA mutations are frequent in metastatic HR⁺ breast cancer. Intra-patient PIK3CA mutational heterogeneity was observed with cases of concordance and discordance when comparing early to advanced disease. The study presents the utilization of a liquid biopsy, thereby paving the way towards the application of a more personalized medicine in the management of patients with metastatic cancer.