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Background: Leukemic stem cells (LSCs) play a central role in relapse and refractory cases of leukemia patients. This cell has been found to resist to a conventional chemotherapy more than leukemic cells. Novel therapeutic strategy directly targets to eliminate the LSCs for eradication of the disease. Abnormal of leukemic cell proliferation is the main problem. The mechanism is involved in many proteins in cell signaling pathway. Wilms’ tumor 1 protein (WT1) is the transcription factor protein. It overexpresses and relates to leukemic cell proliferation but there is no report of WT1 protein expression in the LSCs. Objectives: To compare the percent of LSC (CD34+/CD38-) population and WT1 protein expression levels in KG-1a, KG-1, and K562 cell lines. Materials and methods: Leukemic cells were determined percent of LSC population and WT1 protein level by flow cytometry and Western blot analysis. Results: The result showed that LSC population in KG-1a, KG-1, and K562 cells were 92.82±3.28, 75.95±4.83, and 0.44±0.51%, respectively. Almost cell population (over than 99%) in K562 cells was leukemic blast cells (CD34-). WT1 protein levels by mean fluorescent intensity (MFI) analysis in LSCs of KG-1a, KG-1, and K562 cells were 46.8±5.92, 59.54±4.65, and 183.42±17, respectively. By Western blot analysis, KG-1a cells showed the highest CD34 protein level while KG-1 and K562 cells were 35.68±11.01 and 3.56±3.56%, respectively, when compared to that of KG-1a (100%). Moreover, K562 cells showed the highest WT1 protein level (100%), followed by KG-1 and KG-1a cells with the expression values of 67.23±6.86 and 42.52±5.84%, respectively. Conclusion: WT1 protein expression levels in LSCs of KG-1a cells was less than KG-1 and K562 cells. WT-1 expression is related with the leukemic cell proliferation rate.