Inevitable recurrence after radiochemotherapy is the major problem in the treatment of glioblastoma, the most prevalent type of adult brain malignancy. Glioblastomas are notorious for a high degree of intratumor heterogeneity manifest through a diversity of cell types and molecular patterns. The current paradigm of understanding glioblastoma recurrence is that cytotoxic therapy fails to target effectively glioma stem cells. Recent advances indicate that therapy-driven molecular evolution is a fundamental trait associated with glioblastoma recurrence. There is a growing body of evidence indicating that intratumor heterogeneity, longitudinal changes in molecular biomarkers and specific impacts of glioma stem cells need to be taken into consideration in order to increase the accuracy of molecular diagnostics still relying on readouts obtained from a single tumor specimen. : This study integrates a multisampling strategy, longitudinal approach and complementary transcriptomic investigations in order to identify transcriptomic traits of recurrent glioblastoma in whole-tissue specimens of glioblastoma or glioblastoma stem cells. In this study, 128 tissue samples of 44 tumors including 23 first diagnosed, 19 recurrent and 2 secondary recurrent glioblastomas were analyzed along with 27 primary cultures of glioblastoma stem cells by RNA sequencing. A novel algorithm was used to quantify longitudinal changes in pathway activities and model efficacy of anti-cancer drugs based on gene expression data. : Our study reveals that intratumor heterogeneity of gene expression patterns is a fundamental characteristic of not only newly diagnosed but also recurrent glioblastomas. Evidence is provided that glioblastoma stem cells recapitulate intratumor heterogeneity, longitudinal transcriptomic changes and drug sensitivity patterns associated with the state of recurrence. : Our results provide a transcriptional rationale for the lack of significant therapeutic benefit from temozolomide in patients with recurrent glioblastoma. Our findings imply that the spectrum of potentially effective drugs is likely to differ between newly diagnosed and recurrent glioblastomas and underscore the merits of glioblastoma stem cells as prognostic models for identifying alternative drugs and predicting drug response in recurrent glioblastoma. With the majority of recurrent glioblastomas being inoperable, glioblastoma stem cell models provide the means of compensating for the limited availability of recurrent glioblastoma specimens.