Gliomas are the most common type of primary malignant adult brain tumor. They appear to originate from neuroglial stem or progenitor cells, and are therapeutically challenging due to an invasive growth pattern and the absence of effective therapies. We have analyzed cellular, molecular and proteomic features and defined the therapeutic response profiles of four IDH1-wildtype glioma stem cell (GSC) cultures. All four GSC cultures were established from Grade IV glioblastoma (GBM) surgical resection tissue, can be continuously propagated and are highly enriched for stem/tumor repopulating cells. Integrated genomic and proteomic analysis of all four cultures was performed, together with use of a dual molecular bar-coding strategy to assess GSC population heterogeneity and the response to ionizing radiation. These well-characterized, bar-coded GSC cultures provide an experimentally tractable resource for investigating glioma biology, and to use to identify new and potentially more effective GBM therapies and treatment regimens.