<jats:title>Abstract</jats:title> <jats:p>Anatomical and phytochemical studies of <jats:italic>Zinnia angustifolia</jats:italic> HBK, Z. <jats:italic>elegans</jats:italic> Jacq., and their interspecific hybrids were initiated to define factors affecting ray floret color. Clones of Z. <jats:italic>angustifolia</jats:italic> with ivory, orange, or white ray florets, and Z. <jats:italic>elegans</jats:italic> lines with orange, pink, red, white, or yellow ray florets were crossed intraspecifically to produce S<jats:sub>1</jats:sub> and F<jats:sub>1</jats:sub> progeny. Interspecific hybrids were produced by crossing orange- or white-flowered Z. <jats:italic>angustifolia</jats:italic> clones (as ♀♀) with Z. <jats:italic>elegans</jats:italic> lines (as ♂♂). In both species and interspecific hybrids, pigments were present mainly in the upper epidermis, more dilute in the lower epidermis, and not observed in internal parenchyma. Variation in ray floret color in Z. <jats:italic>elegans</jats:italic> was primarily due to presence or absence of carotenoids in chromoplasts and anthocyanidins in vacuoles. Quantitative differences in the anthocyanidins pelargonidin and cyanidin were detected among Z. <jats:italic>elegans</jats:italic> lines; in vivo expression of anthocyanins was influenced by epidermal cell pH. Ray floret color in Z. <jats:italic>angustifolia</jats:italic> was due to presence or absence of two novel anthocyanidins and ivory-colored flavonoids, with no attributable effect of epidermal cell pH on anthocyanin expression. Carotenoids were not present in ray florets of Z. <jats:italic>angustifolia</jats:italic> × Z. <jats:italic>elegans</jats:italic> hybrids, regardless of whether carotenoids were expressed in the Z. <jats:italic>elegans</jats:italic> parent line. Presence or absence of the two novel anthocyanidins from Z. <jats:italic>angustifolia</jats:italic> and cyanidin from Z. <jats:italic>elegans</jats:italic> permitted unique combinations of floral pigments in interspecific hybrids not previously found in either species.</jats:p>