Glioblastoma multiforme (GBM) also categorized as a grade IV astrocytoma, is an aggressive brain tumor which invades the surrounding brain tissue. Hyperthermia is known to be effective for chemoradiotherapy to sensitize cancer cells to radiation as a treatment option for patients with GBM. The current study was performed in order to assess and compare the properties of the astrocyte and cancer stem cells isolated from glioblastoma exposed to hyperthermia. Astrocytes and cancer stem cells were isolated from human glioblastoma tissue. Glioblastoma tissues were digested and cultured in culture medium supplemented with B27, basic broblast growth factor and epidermal growth factor. The morphology and speci c markers were evaluated in astrocyte and cancer stem cell of human glioblastoma through immunocytochemistry and quantitative real-time RT-PCR. The multipotentiality of cancer stem cells was presented using differentiation potential into neurons, oligodendrocytes, and astrocytes. For hyperthermia, cells were exposed to temperatures at 42-46˚C for 1h using a water bath.Cell survival rate by MTT assay and apoptosis using quantitative real-time RT-PCR and western blot were evaluated. Results demonstrated that there were two morphology types in cell culture including epithelioid morphology and broblastic morphology. Astrocytes were con rmed via expression of the Glial brillary acidic protein (GFAP) protein; whereas, cancer stem cells (CSCs) were round and oating in the culture medium. Immunocytochemical staining indicated that nestin, CD133 and SRY-box 2 (SOX2) antigens were positively expressed in primary neurospheres. Results indicated that cancer stem cells of glioblastoma are multipotent and are able to differentiate into neurons, oligodendrocytes, and astrocytes.The current study obtained evidence via apoptosis evaluation that CSCs are resistant to hyperthermia when compared to astrocytes isolated from glioblastoma. Furthermore, hyperthermia was demonstrated to decrease cell resistance, which may be effective for chemo-radiotherapy to sensitize cancer cells to radiation. Taken together, CSCs of glioblastoma could be used as a powerful tool for evaluating the tumorigenesis process in the brain and developing novel therapies for treatment of GBM.