Multipotent neural crest cells (NCCs) are a major extracardiac component of cardiovascular development. Although recognized as contributing cells to the arterial valves at early developmental stages, NCC persistence in the valves at later times or in the adult heart is controversial. We analyzed NCC persistence and contributions to both semilunar and atrioventricular (AV) valves in the mature heart. Two NCC-specific promoters driving Cre recombinase, Wnt1-Cre and P0-Cre, were mated with floxed reporter mice, R26R or CAG-CAT-EGFP, to map NCC fate. Hearts were analyzed before aorticopulmonary (AP) septation through adult stages. As previously demonstrated, strong NCC labeling was detected in ventral and dorsal outflow cushions before AP septation. In contrast to previous reports, we found that substantial numbers of labeled cells persisted in the semilunar valves in late fetal, neonatal, and adult hearts. Furthermore, NCCs were also found in the AV valves, almost exclusively in the septal leaflets. NCCs in the AV valves expressed melanocytic and neurogenic markers. However, cells labeled in the proximal cardiac conduction system exhibited neurogenic and gliagenic markers, whereas some NCCs expressed no differentiation specific markers. These results suggest that cardiac NCCs contribute to the mature valves and the cardiac conduction system and retain multipotent characteristics late in development.