Technical Abstract: Mammary stem cells (MaSC) account for the cell lineage of mammary epithelia and provide for mammary growth, development and tissue homeostasis. The presence of MaSC was clearly demonstrated by the generation of an entire mammary gland from a single cell implanted into epithelium-ablated mammary fat pads of recipient mice. Attempts to evaluate the molecular character of MaSC have utilized fluorescence-activated cell sorting or the in vitro cultivation of cells from enzymatically dissociated tissue, as means to obtain a population of cells that is enriched for MaSC. However, utilization of these approaches necessitates the loss of histological information encoding the in vivo locale and function of MaSC and their more committed progenitor cell progeny. Here we report the use of an alternative approach, laser microdissection, to excise putative MaSC and control cells from their in situ locations in histological cryosections and to characterize the molecular properties of these cells. We identified potential MaSC in calves based upon their ability to retain bromodeoxyuridine-labeled DNA for an extended period. We then conclude that label retaining epithelial cells in the basal epithelial layer are MaSC, as these cells showed enriched expression of genes that are associated with stem cell attributes; whereas label retaining epithelial cells in suprabasal epithelial layers are progenitor cells, expressing some genes that are associated with stem cell attributes along with those indicative of cell differentiation. Our results also provide a molecular profile and novel candidate markers, confirmed by immunohistochemistry, for bovine MaSC, which have probable application to the study of MaSC in other species.