ABSTRACTThe adult mouse heart possesses an extensive lymphatic plexus draining predominantly the subepicardium and the outer layer of the myocardial wall. However, the development of this plexus has not been entirely explored, partially because of the lack of suitable methods for its visualization as well as prolonged lymphatic vessel formation that starts prenatally and proceeds during postnatal stages. Also, neither the course nor location of collecting vessels draining lymph from the mouse heart have been precisely characterized. In this article, we report that murine cardiac lymphatic plexus development that is limited prenatally only to the subepicardial area, postnatally proceeds from the subepicardium toward the myocardial wall with the base‐to‐apex gradient; this plexus eventually reaches the outer half of the myocardium with a predominant location around branches of coronary arteries and veins. Based on multiple marker immunostaining, the molecular marker‐phenotype of cardiac lymphatic endothelial cells can be characterized as: Prox‐1+, Lyve‐1+, VEGFR3+, Podoplanin+, VEGFR2+, CD144+, Tie2+, CD31+, vWF−, CD34−, CD133−. There are two major collecting vessels: one draining the right and left ventricles along the left conal vein and running upwards to the left side of the pulmonary trunk and further to the nearest lymph nodes (under the aortic arch and near the trachea), and the other one with its major branch running along the left cardiac vein and further on the surface of the coronary sinus and the left atrium to paratracheal lymph nodes. The extracardiac collectors gain the smooth muscle cell layer during late postnatal stages. Anat Rec, 297:1115–1130, 2014. © 2014 Wiley Periodicals, Inc.