Breast cancer is a highly heterogeneous disease at both the molecular and pathological levels. To understand this heterogeneity and ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial hierarchy. Lineage tracing is a key strategy for assessing the stem cell hierarchy as it allows stem and progenitor cell fate to be studied in situ in the context of development, tissue maintenance and disease. We have combined lineage tracing with a novel three-dimensional (3D) imaging strategy to explore the relative contributions of stem and progenitor cells to post-natal mammary gland development and tissue homeostasis. Cell lineage tracing studies also provide the current gold standard for identifying ‘cells of origin’ in cancer. Towards this end, we are utilizing newly generated transgenic strains harbouring lineage-specific gene regulatory regions to direct the expression of specific mammary oncogenic lesions to discrete epithelial cell types. In terms of human breast cancer, the interrogation of molecular profiles has provided insight into potential ‘cells of origin’ in preneoplastic tissue for basal-like cancers arising in BRCA1 mutation carriers. Finally, the identification of deregulated genes/pathways in these crucial target cell populations may enable earlier detection of malignancies and lead to preventative therapies for individuals at high risk of developing breast cancer.