Due to its optical clarity and rudimentary heart structure (i.e., single atrium and ventricle), the zebrafish provides an excellent model for studying the genetic, morphological, and functional basis of normal and pathophysiological heart development in vivo. Recent advances in high-speed confocal imaging have made it possible to capture 2D zebrafish heart wall motions with temporal and spatial resolutions sufficient to characterize the highly dynamic intravital flow-structure environment. We have optimized protocols for introducing fluorescent tracer particles into the zebrafish cardiovasculature, imaging intravital heart wall motion, and performing high-resolution blood flow mapping that will be broadly useful in elucidating flow-structure relationships.