Parity (childbearing) significantly decreases a woman’s risk of breast cancer. Several factors within the mammary gland are postulated to contribute to parity-induced protection including (but not restricted to), a reduction in the number of mammary stem cells (MaSCs). We tested this in our mouse model of parity using two currently available MaSC isolation protocols; CD24+CD49fhi and CD24+ Sca-1neg. We found a reduction in MaSC in parous animals compared to nulliparous (no offspring) animals only when using the CD24+CD49fhi isolation strategy. Direct comparison of the stem cell activity of the two isolation strategies revealed that the CD24+CD49fhi population is 13-fold more enriched for stem cell activity using the gold standard mammary fat pad transplant assay. We wished to investigate the difference in MaSC enrichment, postulating that exclusion of Sca-1 positive cells may account for the reduced MaSC enrichment within the CD24+Sca-1neg population. Direct comparison of the repopulating potential of the CD24+CD49fhiSca-1- and CD24+CD49fhiSca-1+ populations revealed that Sca-1 positivity in addition to CD24+CD49fhi does not increase stem cell enrichment. Whilst the CD24+CD49fhiSca-1- population did show greater repopulating potential than the CD24+CD49fhiSca-1+ population, Sca-1 negativity did not result in greater enrichment for MaSC activity than using CD24+CD49fhi alone. Additionally, whilst we identified that CD24+CD49fhiSca-1+ cells were increased at younger ages (when stem cells are presumed abundant) compared to CD24+CD49fhiSca-1- cells, we found that both populations were actively cycling, rather than residing in a state of dormancy, typical of stem cells. These results indicate that Sca-1 cannot be used to enrich for MaSC cells in combination with the current optimal MaSC isolation strategy, that is CD24+ and CD49fhi expression.