Purpose We aimed to compare spatial extent of high-grade subregions detected with combined [F-18]-dihydroxyphenylalanine (F-18-DOPA) PET and MRI to the one provided by advanced multimodal MRI alone including Contrast-enhanced (CE) and Perfusion weighted imaging (PWI). Then, we compared the accuracy between imaging modalities, in a per biopsy analysis. Methods Participants with suspected diffuse glioma were prospectively included between June 2018 and September 2019. Volumes of high-grade subregions were delineated respectively on F-18-DOPA PET and MRI (CE and PWI). Up to three per-surgical neuronavigation-guided biopsies were performed per patient. Results Thirty-eight biopsy samples from sixteen participants were analyzed. Six participants (38%) had grade IV IDH wild-type glioblastoma, six (38%) had grade III IDH-mutated astrocytoma and four (24%) had grade II IDH-mutated gliomas. Three patients had intratumoral heterogeneity with coexisting high- and low-grade tumor subregions. High-grade volumes determined with combined F-18-DOPA PET/MRI (median of 1.7 [interquartile range (IQR) 0.0, 19.1] mL) were larger than with multimodal MRI alone (median 1.3 [IQR 0.0, 12.8] mL) with low overlap (median Dice's coefficient 0.24 [IQR 0.08, 0.59]). Delineation volumes were substantially increased in five (31%) patients. In a per biopsy analysis, combined F-18-DOPA PET/MRI detected high-grade subregions with an accuracy of 58% compared to 42% (p = 0.03) with CE MRI alone and 50% (p = 0.25) using multimodal MRI (CE + PWI). Conclusions The addition of F-18-DOPA PET to multimodal MRI (CE and PWI) enlarged the delineation volumes and enhanced overall accuracy for detection of high-grade subregions. Thus, combining F-18-DOPA with advanced MRI may improve treatment planning in newly diagnosed gliomas.