AbstractAdult bovine mammary stem cells possess the ability to regenerate in vivo clonal outgrowths that mimic functional alveoli. Commonly available techniques that involve immunophenotype-based cell sorting yield cell fractions that are moderately enriched, far from being highly purified. Primary bovine mammary epithelial cells segregated in four different populations according to the expression of P-Cadherin and CD49f. Sorted cells from each fraction were tested for the presence of lineage-restricted progenitors and stem cells. Only cells from the CD49f^high/P-Cadherin^neg subpopulation were able to give rise to both luminal- and myoepithelial-restricted colonies in vitro and generate organized outgrowths in vivo, which are hallmarks of stem cell activity. After whole transcriptome analysis, we found gene clusters to be differentially enriched that relate to cell-to-cell communication, metabolic processes, proliferation, migration and morphogenesis. When we analyzed only the genes that were differentially expressed in the stem cell enriched fraction, clusters of downregulated genes were related to proliferation, while among the upregulated expression, cluster of genes related to cell adhesion, migration and cytoskeleton organization were observed. Our results show that P-Cadherin separates mammary subpopulations differentially in progenitor cells or mammary stem cells. Further we provide a comprehensive observation of the gene expression differences among these cell populations which reinforces the assumption that bovine mammary stem cells are typically quiescent.