BACKGROUND: Tumor hypoxia and proliferation are independent predictors of poor prognosis in glioblastoma and WHO grade 4 IDH-mutant astrocytoma, and are closely linked and can synergistically contribute to local recurrence (LR) and poor overall survival (OS). These two hallmarks can be imaged using FMISO and FLT PET, but only on different days due to the PET intrinsic limitation, which jeopardizes the clinical feasibility and accuracy of multi-parametric studies. PURPOSE: In this study, we assess the feasibility of dual-[FMISO+FLT]-PET in a cohort of patients with glioblastoma and WHO grade 4 IDH-mutant astrocytoma. METHODS: Eight patients underwent 90 min dynamic PET (dynPET) with staggered FMISO/FLT injections followed by two 10 min scans at 120 and 180 min post-FMISO injection, respectively. The target volume (TV) was delineated on the 180-min imageset. The FMISO input function (IF) was derived from dynPET images of the carotids using the first 50 min, and then extrapolated to the rest of dynPET using a 3-exp fit. The IF_FLT was deduced by subtracting the IF_FMISO from IF_FMISO+FLT over the range > 50 min. The FMISO and FLT kinetic rate constants (KRCs) of the TV and cerebellar cortex (reference tissue) were estimated using kinetic modeling (KM) with a parallel dual-1-tissue-2-compartment model. RESULTS: Seven out of eight patients with a total of 13 lesions completed the study. All lesions were [FMISO+FLT]-avid at 180 min post-FMISO injection with a mean SUVR of 1.72 (range:1.26-3.23). IDH-mutant WHO grade 4 astrocytomas showed reduced tumor hypoxia. Mean lesion KRCs were K_1-FMISO = 0.18 mL/cc/min (range:0.042-0.432), k_i-FMISO = 0.011 min^-1 (range:0.00-0.039), K_1-FLT = 0.103 mL/cc/min (range: 0.004-0.357), and K_i-FLT = 0.014 mL/min/g (range: 0.00-0.062). Cerebellar cortex KRCs were K_1-FMISO = 0.098 mL/cc/min (range:0.055-0.225), k_i-FMISO = 0.008 min^-1 (range:0.002-0.014), K_1-FLT = 0.089 mL/cc/min (range: 0.001-0.299), and K_i-FLT = 0.003 mL/min/g (range:0.00-0.007). Lesion perfusion and hypoxia were inversely correlated (R = 0.99). CONCLUSIONS: Dual-[FMISO+FLT]-PET can provide detailed characterization of tumor microenvironment and interaction of multiple hallmarks that yield radio-resistance. This can improve the accuracy of image-guided radiosurgery and radiotherapy, thereby improving clinical outcomes in patients with glioblastoma and IDH-mutant WHO grade 4 astrocytoma.