© 2019 The Authors Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133 + PN signature, transition to a CD109 + MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133 − /CD109 + cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.