During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/− or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH−CD44+CD24− human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24− by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity. •Isolation and RNA-seq of ALDH+ and CD44+CD24− breast cells•Unlike in cancer, there is substantial overlap in ALDH+ and CD44+CD24− populations•Single-cell analysis of ALDH+ cells identifies unexpected subpopulation structure•Hybrid epithelial/mesenchymal ALDH+ cells have a cancer-like expression signature In this article, Colacino and colleagues use flow-cytometry-sorted populations and single-cell analyses to investigate human mammary stem cells. They discover unexpected phenotypic and functional heterogeneity at the single-cell level, including a subpopulation of ALDH+ stem cells with a hybrid epithelial/mesenchymal phenotype and triple-negative breast cancer-like gene expression pattern.