The goal of this study is to evaluate a new 68Ga-based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [68Ga]-HP-DO3A-nitroimidazole ([68Ga]-HP-DO3A-NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP-DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [68Ga]-HP-DO3A-NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O2. The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 hrs post-injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [68Ga]-HP-DO3A-NI displayed very favorable pharmacokinetic properties as it was cleared largely through the kidneys with little to no accumulation in liver, heart, or lung (% ID/g < 0.5%) at 2 hours p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound [68Ga]-HP-DO3A, which lacks the nitroimidazole moeity, and by PET imaging of tumor-bearing mice breathing air versus 100% O2. Given the commercial availability of cGMP 68Ge/68Ga generators and the ease of 68Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy.