Abstract Purpose: Micro-computed tomography (micro-CT) scan provides high-resolution three-dimensional images of mineralized tissues in small animal models. Contrast enhancement is essential to visualize non-mineralized tissues with micro-CT scan. We attempted to compare the two most common contrast agents to stain and image mouse cardiac structures. Approach: Ex-vivo micro-CT scan images of the mouse hearts were obtained following staining by potassium iodide or phosphotungstic acid (PTA). PTA-stained samples were imaged after various durations following staining (14 days, 25 days, 187 days, and 780 days), whereas iodine-stained samples were imaged after 72 hours. We compared median staining intensity between PTA and iodine at 0.1-mm intervals from the edge using the Mann Whitney test with correction for multiple comparisons. Results: Sixty post-natal mice hearts were stained with either PTA or iodine and imaged using micro-CT scan. Iodine proved to be faster and more uniform in complete enhancement of cardiac tissue in as short as 72 h, whereas PTA required a significantly longer time period to penetrate mouse cardiac structure (>150 days). Median staining intensity with iodine was strongly higher than that with PTA from 0.1-to 1.5-mm distance from the epicardial edge (2-tailed P value