The isolation and characterisation of mammary stem cells is an important step towards elucidating the hierarchy of epithelial cell development in the mammary gland and identifying cells that are targets of breast carcinogenesis. Mammary stem cells have recently been prospectively isolated through the identification of specific cell surface markers and in vivo transplantation into cleared fat pads. These cells were demonstrated to reconstitute an entire mammary gland comprising all mature epithelial cell types and to be capable of self-renewal on serial transplantation, thus possessing the defining features of stem cells. Notably, mouse mammary stem cells were found to share the hallmark properties of the basal subtype of breast cancer. This review will summarize the strategy used in the identification of mouse mammary stem cells and their characterisation.