Hypoxia has been identified as a major adverse prognostic factor for tumor progression and for resistance to anticancer treatment. Various approaches have been evaluated to assess tumor hypoxia in vivo. PET imaging in particular has emerged as a promising non-invasive tool to accurately characterize tumor oxygenation, thus offering the potential to optimize and individualize a therapy for patients suffering from cancer. The current PET tracers and animal models for the study of tissue hypoxia including aspects of small animal PET imaging are reviewed in this paper. Special emphasis is placed on human PET studies assessing tumor hypoxia in patients with different tumor entities, various anticancer therapies (chemotherapy, radiation therapy, hypoxia targeting chemotherapy) as well as studies deriving prognostic factors and outcome data. Methodological advantages underpinning PET/CT in the imaging of hypoxia are discussed. The great promise of PET/CT is its potential as a single imaging modality for whole body staging providing both anatomical and biological information on the tumor as a whole. It allows a more precise estimation of the hypoxic tumor volume as well as comparisons on a voxel-by-voxel basis (parametric mapping). Finally, PET and PET/CT are discussed in the framework of individualized therapies with its special significance for intensity modulated radiation therapy and selective dose escalation in hypoxic tumor regions as well as more systemic approaches such as hypoxia-directed cytotoxins.