Quantification of tumour R2* and oxygen-induced ΔR2* and ΔR1 are being investigated as potential biomarkers of tumour hypoxia, but their relationship is complex and not well understood. Here, we used a validated R1 biomarker (oxygen refractory fraction, termed “Oxy-R”) to segment tumours into hypoxic and non-hypoxic sub-regions. This revealed a clear relationship between hypoxic status and native R2* and hyperoxia-induced ΔR2*. Preclinical findings were replicated in clinical data from patients with renal carcinoma. These data highlight the importance of heterogeneity-based analysis of tumours and provide further validation of Oxy-R as a biomarker of tumour hypoxia.