Neural crest cells constitute a multipotent cell population that gives rise to diverse cell lineages. The neural crest arising from the postotic hindbrain is known as the 'cardiac' neural crest, and contributes to the great vessels and outflow tract endocardial cushions, but the neural crest contribution to structures within the heart remains largely controversial. Here we demonstrate that neural crest cells from the preotic region migrate into the heart and differentiate into coronary artery smooth muscle cells. Preotic neural crest cells preferentially distribute to the conotruncal region and interventricular septum. Ablation of the preotic neural crest causes abnormalities in coronary septal branch and orifice formation. Mice and chicks lacking endothelin signalling show similar abnormalities in the coronary artery, indicating its involvement in neural crest-dependent coronary artery formation. This is the first report that reveals the preotic neural crest contribution to heart development and smooth muscle heterogeneity within a coronary artery.