Radioresistance is the main determinant of treatment outcome in head and neck cancer. The aim of this study was to establish radioresistant head and neck cancer cell lines and isolate cancer stem cells from them to investigate the role of cancer stem cells in radioresistant head and neck cancer.To induce radioresistant cell lines, radiation was delivered to SCC15, SCC25, and QLL1 cells with an accumulated dosage of 60Gy over 30 cycles of irradiation. After a total of 60Gy of irradiation, the radioresistance of irradiated cancer cells was verified by MTT assay. The radioresistant cells were cultured in serum-free medium in ultra-low-attachment culture flasks to induce sphere-forming cells. Then, sphere-forming cells were analyzed using Western blotting to identify the expression of stem cell markers, such as Nanog and Sox-2.The MTT assay of cell viability showed more radioresistance in the irradiated cancer cell lines than in the non-irradiated cancer cell lines. Sphere-forming cells were identified in all three cancer cell lines 3–5 days after serum deprivation. All sphere-forming cells from the three cancer cell lines expressed stem cell markers. Sphere-forming cells showed more radioresistance than monolayer cells after irradiation by colony forming assay.Cancer stem cells seem to play an important role in the radioresistance of head and neck cancer. Further research is required to find a precise mechanism of radioresistance related to cancer stem cells.