Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity resulting in targeted therapy resistance and other challenges that make the management and treatment of these diseases rather difficult. Due to the presence of admixtures of non-neoplastic cells with polyclonal cell populations, it is difficult to define cancer genomes in patient samples. By isolating tumor cells from normal cells, and enriching distinct clonal populations, clinically relevant genomic aberrations that drive disease can be identified in patients in vivo. An in-depth analysis of clonal architecture and tumor heterogeneity was performed in a stage II chemoradiation-naïve breast cancer from a sixty-five year old patient. DAPI-based DNA content measurements and DNA content-based flow sorting was used to to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes of multiple biopsies from the breast cancer. The detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present within the tumor. The clonal analysis revealed driver events for tumor progression (a heterozygous germline BRCA2 mutation converted to homozygosity within the tumor by a copy number event and the constitutive activation of Notch and Akt signaling pathways. The highlighted approach has broad implications in the study of tumor heterogeneity by providing a unique ultra-high resolution of polyclonal tumors that can advance effective therapies and clinical management of patients with this disease.