Background Histologic studies have demonstrated that lipid content and its spatial distribution is related to plaque vulnerability. However, in vivo imaging is still limited. Photoacoustic imaging may provide novel in vivo insights into these lipid-rich plaques. Objectives This study sought to examine whether intravascular photoacoustic tomography (IVPAT) allows localization and quantification of lipid content in atherosclerotic plaques. Methods Rabbits fed with a high-fat/high-cholesterol diet served as the atherosclerotic model. Catheter-based IVPAT was used to evaluate pixel-based lipid relative concentration (LRC) of the vessel wall. The aorta of 4 groups of rabbits (n = 12) were examined ex vivo with IVPAT after 0, 5, 10, and 15 weeks of a high-fat diet, respectively. Six rabbits underwent 3-dimensional (3D) IVPAT after 20 weeks of the high-fat diet. Three rabbits were examined in vivo using IVPAT without interruption of blood flow. Concentration-based lipid map and quantitative index were calculated. For subsequent histologic correlation, all specimens were evaluated with Oil Red O staining. Results Cross-sectional LRC maps allowed visualization of concentration and depth information of lipid content in the atherosclerotic plaques. Lipid accumulation within plaque, assessed by the maximum LRC, mean LRC, and high lipid content area correlated to duration of a high-fat diet. Three-dimensional LRC maps enabled overall evaluation of focal plaques in an intact explanted aorta including spatial and structural features. In vivo-obtained LRC maps accurately showed the structure of lipid core with high contrast. Ex vivo and in vivo IVPAT results were highly consistent with histological results. Conclusions In an animal model, IVPAT allowed characterization of spatial and quantitative features of lipid-rich plaques.