Identification and isolation of breast cancer stem cells (CSCs) based on CD44/CD24 expression and/or enzymatic activity of aldehyde dehydrogenase 1 (ALDH1). However, the differences among the CD44+/CD24‑/low cells, ALDH1+ cells and the overlap between the sub‑populations have not been frequently investigated. Thus, it is imperative to improve the understanding of breast CSC with different stem markers. CD44+/CD24‑/low, ALDH1+ and ALDH1+CD44+/CD24‑/low cell populations were isolated from fresh breast cancer tissues and analyzed by flow cytometry and immunofluorescence. Mammosphere formation, cell proliferation assay and Transwell experiments, were used to analyze self‑renewal, proliferation and invasion, respectively, for each sub‑population. Finally, in vivo experimentation in mice was performed to evaluate the tumorigenic abilities of the sub‑populations. The sub‑populations of CD44+/CD24‑/low, ALDH1+ and ALDH1+CD44+/CD24‑/low in human breast cancer cells, represented the 7.2, 4.6 and 1.5% of the total tumor cell population, respectively. ALDH1+CD44+/CD24‑/low cells had the strongest ability of self‑renewal, invasion, proliferation and tumorigenicity compared with the other sub‑populations (P<0.05). In conclusion, different phenotypes of CD44+/CD24‑/low, ALDH1+ and ALDH1+CD44+/CD24‑/low were isolated and demonstrated that breast CSCs are heterogeneous, and they exhibit distinct biological characteristics. As ALDH1+CD44+/CD24‑/low cells demonstrated the strongest stem‑like properties, it may be a useful specific stem cell marker. The utilization of more reliable biomarkers to distinguish the breast CSC pool will be important for the development of specific target therapies for breast cancer.