Abnormalities in valvuloseptal development significantly contribute to congenital heart defects, yet the underlying causes are complex and poorly understood. Early cardiac regulatory genes are differentially expressed during valvuloseptal development, consistent with novel functions during heart chamber formation in chicken and mouse embryos. Distinct valve cell lineages were identified in the leaflets, chordae tendineae, and myotendinous junctions with the papillary muscles based on restricted expression of extracellular matrix molecules. Specific cell types within these structures demonstrate characteristics of chondrogenesis and tendon development, identified by scleraxis, type II collagen, and tenascin expression. In chicken embryos, valve remodeling and maturation accompanies a decrease in mitotic index indicated by reduced bromodeoxyuridine incorporation. Analysis of Tie2‐cre × ROSA26R mice demonstrates that mature valve structures, including the atrioventricular and outflow tract semilunar valve leaflets, chordae tendineae, and the fibrous continuity that connects the septal leaflets of mitral and tricuspid valves, arise from endothelial cells of the endocardial cushions. Together, these studies provide novel insights into the origins and cell lineage diversity of mature valve structures in the developing vertebrate heart. Developmental Dynamics 230:239–250, 2004. © 2004 Wiley‐Liss, Inc.