The partial pressure in oxygen remains challenging to map in the brain. Two main strategies exist to obtain surrogate measures of tissue oxygenation: the tissue saturation studied by magnetic resonance imaging (S_tO₂-MRI) and the identification of hypoxia by a positron emission tomography (PET) biomarker with 3-[¹⁸F]fluoro-1-(2-nitro-1-imidazolyl)-2-propanol ([¹⁸F]-FMISO) as the leading radiopharmaceutical. Nonetheless, a formal validation of S_tO₂-MRI against FMISO-PET has not been performed. The objective of our studies was to compare the two approaches in (a) the normal rat brain when the rats were submitted to hypoxemia; (b) animals implanted with four tumour types differentiated by their oxygenation. Rats were submitted to normoxic and hypoxemic conditions. For the brain tumour experiments, U87-MG, U251-MG, 9L and C6 glioma cells were orthotopically inoculated in rats. For both experiments, S_tO₂-MRI and [¹⁸F]-FMISO PET were performed sequentially. Under hypoxemia conditions, S_tO₂-MRI revealed a decrease in oxygen saturation in the brain. Nonetheless, [¹⁸F]-FMISO PET, pimonidazole immunohistochemistry and molecular biology were insensitive to hypoxia. Within the context of tumours, S_tO₂-MRI was able to detect hypoxia in the hypoxic models, mimicking [¹⁸F]-FMISO PET with high sensitivity/specificity. Altogether, our data clearly support that, in brain pathologies, S_tO₂-MRI could be a robust and specific imaging biomarker to assess hypoxia.