Glioblastoma therapy relies on the alkylating drug temozolomide (TMZ) administered to irradiated patients post-neurosurgery, which increases overall survival but cannot prevent fatal disease relapse. Using clinical samples and glioblastoma models, we here identify TMZ-driven enrichment of ALDH1A1+ tumor subclones acquiring AKT-dependent drug resistance en route to relapse. We demonstrate that this recurrent phenotype switch is predictable and can be countered by a sequential rather than simultaneous combinatorial treatment approach. Overall design: Microarray gene expression was conducted on paired (treatment-naive and relapse) patient-derived and experimentally generated IDH wt glioblastoma samples.