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.