Using the transposon-mediated enhancer trap (ET), we generated 18 cardiac enhancer trap (CET) transgenic zebrafish lines. They exhibit EGFP expression in defined cell types—the endocardium, myocardium, and epicardium—or in anatomical regions of the heart—the atrium, ventricle, valves, or bulbus arteriosus. Most of these expression domains are maintained into adulthood. The genomic locations of the transposon insertions were determined by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The expression pattern of EGFP in some CETs is unique and recapitulates expression of genes flanking the transposon insertion site. The CETs enabled us to capture the dynamics of the embryonic heart beating in vivo using fast scanning confocal microscopy coupled with image reconstruction, producing three-dimensional movies in time (4D) illustrating region-specific features of heart contraction. This collection of CET lines represents a toolbox of markers for in vivo studies of heart development, physiology, and drug screening. Developmental Dynamics 239:914–926, 2010. © 2010 Wiley-Liss, Inc.