Tumor hypoxia may play a fundamental role in determining the radiotherapy outcome for several cancer types. Functional imaging with hypoxia specific radiotracers offers a way to visualize and quantify regions of increased radioresistance, which may benefit from dose escalation strategies. Conversion of the uptake in positron emission tomography (PET) images into oxygenation maps offers a way to quantitatively characterize the microenvironment. However, normalization of the uptake with respect to a well-oxygenated reference volume (WOV), which should be properly selected, is necessary when using conversion functions. This study aims at assessing the sensitivity of quantifying tumor oxygenation based on 18F-fluoromisonidazole (FMISO) PET with respect to the choice of the location and the oxygenation level of the WOV in head and neck cancer patients. WOVs varying not only in shape and location but also with respect to the assigned pO2 level were considered. pO2 values other than the standard 60 mmHg were selected according to the specific tissue type included in the volume. For comparison, the volume which would be considered as hypoxic based on a tissue-to-muscle ratio equal to 1.4 was also delineated, as conventionally done in clinical practice. Hypoxia mapping strategies are found highly sensitive to selection of the location of well-oxygenated region, but also on its assigned oxygenation level, which is crucial for hypoxia-guided adaptive dose escalation strategies.