Background Mapping the hypoxic brain in acute ischemic stroke has considerable potential for both diagnosis and treatment monitoring. PET using ¹⁸F-fluoro-misonidazole (FMISO) is the reference method; however, it lacks clinical accessibility and involves radiation exposure. MR-based T2′ mapping may identify tissue hypoxia and holds clinical potential. However, its validation against FMISO imaging is lacking. Here we implemented back-to-back FMISO-PET and T2′ MR in rodents subjected to acute middle cerebral artery occlusion. For direct clinical relevance, regions of interest delineating reduced T2′ signal areas were manually drawn. Methods Wistar rats were subjected to filament middle cerebral artery occlusion, immediately followed by intravenous FMISO injection. Multi-echo T2 and T2* sequences were acquired twice during FMISO brain uptake, interleaved with diffusion-weighted imaging. Perfusion-weighted MR was also acquired whenever feasible. Immediately following MR, PET data reflecting the history of FMISO brain uptake during MR acquisition were acquired. T2′ maps were generated voxel-wise from T2 and T2*. Two raters independently drew T2′ lesion regions of interest. FMISO uptake and perfusion data were obtained within T2′ consensus regions of interest, and their overlap with the automatically generated FMISO lesion and apparent diffusion coefficient lesion regions of interest was computed. Results As predicted, consensus T2′ lesion regions of interest exhibited high FMISO uptake as well as substantial overlap with the FMISO lesion and significant hypoperfusion, but only small overlap with the apparent diffusion coefficient lesion. Overlap of the T2′ lesion regions of interest between the two raters was ∼50%. Conclusions This study provides formal validation of T2′ to map non-core hypoxic tissue in acute stroke. T2′ lesion delineation reproducibility was suboptimal, reflecting unclear lesion borders.