BACKGROUND. Glioblastoma (GB) gold standard treatment combines maximally-safe surgical resection of the contrast-enhanced (CE) central tumor area, as defined by MRI, and chemo-radiotherapy. However, most patients relapse within one year in non-CE peritumoral FLAIR regions. Spectroscopy MRI (MRSI) can discriminate metabolic tumor areas with higher recurrence potential. We showed that regions with Choline/N-acetyl-aspartate index >2 (CNI+) were predictive of relapse sites post-radiotherapy in CE and FLAIR areas. As relapses are mainly imputed to a subpopulation of aggressive and resistant tumor stem-like cells, called GB-initiating cells (GIC), this suggests that CNI+ areas might be enriched in GIC. METHODS. We conducted a prospective trial in 16 eligible GB patients subjected to preoperative MRSI/MRI and subsequent surgery/chemo-radiotherapy to investigate GIC enrichment of CNI+ versus CNI- areas, based on biopsies in CE and FLAIR. We combined in vitro/vivo biological characterization of biopsies and derived GIC lines with biopsy RNAseq analyses. RESULTS. Biopsy characterization by FACS and RNAseq revealed that FLAIR/CNI+ areas showed an enrichment in GIC-population and in stem-related gene signature, but also in pathways related to DNA repair, adhesion/migration and mitochondrial bioenergetics. More, FLAIR/CNI+ samples gave rise to GIC-enriched neurospheres faster than CNI- counterparts. Finally, parameters assessing Biopsy GIC Content and Time to Neurosphere formation in FLAIR/CNI+ areas were associated with worse patient outcome. CONCLUSION. Preoperative MRI/MRSI combination would certainly allow better resection and targeting of CNI+ areas in FLAIR, as their GIC-enrichment can predict worse outcome in GB patients. TRIAL REGISTRATION. ClinicalTrials.govNCT01872221. FUNDING. RITC (RECF1929), GRICR and Plan Cancer 2016 (HTE).