© 2016 Elsevier Inc. Introduction Cardiovascular disease is the leading cause of death in the United States. The identification of vulnerable plaque at risk of rupture has been a major focus of research. Hypoxia has been identified as a potential factor in the formation of vulnerable plaque, and it is clear that decreased oxygen plays a role in the development of plaque angiogenesis leading to plaque destabilization. The purpose of this study is to demonstrate the feasibility of copper-64 labeled diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), a positron-emitting radiopharmaceutical taken up in low-oxygen-tension cells, for the identification of hypoxic and potentially unstable atherosclerotic plaque in a mouse model. Methods 64Cu-ATSM PET was performed in 21 atherosclerotic apolipoprotein E knockout (ApoE−/−) mice, 6 of which were fed high-fat diet (HFD) while the others received standard-chow diet (SCD), and 13 control wild type mice fed SCD. 4 SCD ApoE−/− mice and 4 SCD wild type mice also underwent 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) imaging one day prior to 64Cu-ATSM PET. Results 64Cu-ATSM uptake was increased in the aortic arch in SCD ApoE−/− mice (average aortic arch/muscle (A/M) standardized uptake value ratio 7.5–30 min post injection: (5.66 ± 0.23) compared to control mice (A/M SUV ratio 7.5–30 min post injection (3.87 ± 0.22), p < 0.0001). HFD ApoE−/− mice also showed similarly increased aortic arch uptake on PET imaging in comparison to control mice. Immunohistochemistry in both HFD and SCD ApoE−/− mice revealed noticeable hypoxia by pimonidazole stain in atherosclerosis which was co-localized to macrophage by CD68 staining. Autoradiography assessment demonstrated the presence of hypoxia by 64Cu-ATSM uptake correlated with pimonidazole uptake within the ex vivo atherosclerotic aortic arch specimens. A significant increase in 18F-FDG uptake in the SCD ApoE−/− mice in comparison to controls was also observed at delayed time points. Conclusion This pre-clinical study suggests that 64Cu-ATSM is a potential PET tracer for hypoxia imaging in atherosclerosis. Advances in Knowledge and Implications for Patient Care While studies in humans are necessary for conclusive data, in the long term, a 64Cu-ATSM PET imaging strategy could help facilitate the study of plaque biology in human patients.