Rationale: The spatial distribution of blood flow in hearts of genetically modified mice is a phenotype of interest as derangements in blood flow may precede detectable changes in organ function. However, quantifying the regional distribution of blood flow within organs of mice is challenging because of the small organ volume and the high resolution required to observe spatial differences in flow. Traditional microsphere methods in which the numbers of microspheres per region are indirectly estimated from radioactive counts or extracted fluorescence have been limited to larger organs for two reasons; to assure statistical confidence in the measured flow per region and to be able to physically dissect the organ to acquire spatial information. Objective: We sought to develop methods to quantify and statistically compare the spatial distribution of blood flow within organs of mice. Methods and Results: We developed and validated statistical methods to compare blood flow between regions and to the same regions over time using 15 microm fluorescent microspheres. We then tested this approach by injecting fluorescent microspheres into isolated perfused mouse hearts, determining the spatial location of every microsphere in the hearts and then visualizing regional flow patterns. We demonstrated application of these statistical and visualizing methods in a coronary artery ligation model in mice. Conclusions: These new methods provide tools to investigate the spatial and temporal changes in blood flow within organs of mice at a much higher spatial resolution than currently available by other methods.