Objective: Cancer is a disease in which cells proliferate out of control and in malignancy lead to metastasis. Brain tumor is an example of these cancers. Timely recognition of cancer in its early stages increases the likelihood of treatment. Therefore, the identification of tumor initiating cells is accordingly important and with identifying these cells, their markers and involved signaling pathways, it would be possible to target these cells directly or indirectly and inhibit them with the least side effects for patients. As example of common markers in identifying brain cancer stem cells, CD133, Integrinα6, nestin and L1CAM (as glioma stem cell markers) and OCT4 (as a stemness marker) can be mentioned. These markers are expressed in normal cells in specific levels whereas they over express in brain cancer stem cells. They are recognized by analyzing of their expression level. Therefore in this study, we identify brain cancer stem cells, characterize and isolate them. Materials and Methods: To achieve aims of this study we use variety of techniques. First, brain tumors were recognized and separated by surgeon; they were cultured in proper medium and produce neurosphere. Neurospheres were studied by neurosphere assay method and different passages of them were used for techniques such as immunocytochemistry, flow cytometry and Real time PCR to study of qualitative and quantitative expression of cellular markers. Results: As a result of experiments, we concluded that increase of the listed markers related to tumor nature and stemness potential of these cells. Conclusion: So, we can isolate and characterize cancer stem cells according to gene expression pattern and use them in research and clinical studies for improving the treatment method of CNS tumors.