Radiobiological research over the past decades has provided evidence that cancer stem cell content and the intrinsic radiosensitivity of cancer stem cells varies between tumours, thereby affecting their radiocurability. Translation of this knowledge into predictive tests for the clinic has so far been hampered by the lack of methods to discriminate between stem cells and non-stem cells. New technologies allow isolation of cells expressing specific surface markers that are differentially expressed in tumour cell subpopulations that are enriched for cancer stem cells. Combining these techniques with functional radiobiological assays holds the potential to elucidate the role of cancer stem cells in radioresistance in individual tumours, and to use this knowledge for the development of predictive markers for optimization of radiotherapy.