Cells with high CD44 but low CD24 expression (CD44high/CD24-/low) and high aldehyde dehydrogenase activity (ALDHbr) are widely considered to be drivers of metastasis, therapy resistance and tumor recurrence in breast cancer. However, the role of the CD44high/CD24-/low and ALDHbr phenotypes in identifying tumorigenic cells in breast cancer remains controversial due to the discrepancy in their distribution and tumorigenic potential in intrinsic breast cancer subtypes. In this study, we analyzed the cells expressing these markers in six different breast cancer cell lines representing major breast cancer subtypes (T47D, MCF-7, BT-474, AU-565, Hs578T and MDA-MB-231). CD44high/CD24-/low, ALDHbr and CD44-/low/CD24-/low cell populations were isolated by flow cytometry and analyzed for hallmark stem cell characteristics of differentiation, migration, invasiveness and metastasis using in vitro and in vivo techniques. Our results demonstrate that the CD44-/low/CD24-/low cell population, which is enriched in luminal cell lines (T47D, MCF-7 and BT-474), possesses metastatic and tumorigenic properties. We also show that, contrary to previous claims, the expression of the ALDH1 isoform ALDH1A1 does not affect the tumorigenic potential of cell lines with high ALDH activity (BT-474 and AU-565). Further transcriptomic and clinical studies are needed to determine the potential of these markers as early diagnostic tools and treatment targets.