High-frequency ultrasound biomicroscopy (UBM) has recently emerged as a high-resolution means of phenotyping genetically altered mice and has great potential to evaluate the cardiac morphology and hemodynamics of mouse mutants. However, there is no standard procedure of in vivo transthoracic cardiac imaging using UBM to comprehensively phenotype the adult mice. In this paper, the characteristic mouse thoracic anatomy is elucidated using magnetic resonance (MR) imaging on fixed mice. Besides the left parasternal and apical windows commonly used for transthoracic ultrasound cardiac imaging, a very useful right parasternal window is found. We present strategies for optimal visualization using UBM of key cardiac structures including: 1) the right atrial inflow channels such as the right superior vena cava; 2) the right ventricular inflow tract via the tricuspid orifice; 3) the right ventricular outflow tract to the main pulmonary artery; 4) the left atrial inflow channel, e.g., pulmonary vein; 5) the left ventricular inflow tract via the mitral orifice; 6) the left ventricular outflow tract to the ascending aorta; 7) the left coronary artery; and 8) the aortic arch and associated branches. Two-dimensional ultrasound images of these cardiac regions are correlated to similar sections in the three-dimensional MR data set to verify anatomical details of the in vivo UBM imaging. Dimensions of the left ventricle and ascending aorta are measured by M-mode. Flow velocities are recorded using Doppler at six representative intracardiac locations: right superior vena cava, tricuspid orifice, main pulmonary artery, pulmonary vein, mitral orifice, and ascending aorta. The methodologies and baseline measurements of inbred mice provide a useful guide for investigators applying the high-frequency ultrasound imaging to mouse cardiac phenotyping.