Carotenoids are indispensable for both human health and plant survival. Citrus, is one of the fruit crops richest in carotenoid compounds, with approximately 115 kinds of carotenoids;tremendous diversity in carotenoids composition and concentration exists among various species, showing different colors from nearly white to crimson. The carotenoid biosynthetic pathway and the key carotenogenic genes have been identi-fied in citrus;however, the underlying regulatory mechanisms remain unclear. In this study, among the main species of genus Citrus (primitive, wild, and cultivated), we detected carotenoids in flavedo using High-Performance Liquid Chromatography, and analyzed variations in cis-acting elements in the promoters of key carotenoid pathway genes. Intriguingly, both carotenoid composition and content were generally increased during the evolution of citrus, and the corresponding variations in the promoters were identified, including the gain or loss of critical environ-mental stress-responsive elements and hormone-responsive elements, which are closely associated with carotenoid enhancement. In addition, pummelo has the most heat-responsive elements, but the Mangshan mandarin does not have this element in the promoters of PSY, which is highly related to their geographical origin and indicate that temperature is a critical environmental signal influencing carotenoid accumulation. Moreover, the abscisic acid-responsive motif was rich in almost all the seven species, but the ethylene-responsive motif was deficient, which demystified the unique phytohormone regulation mechanism of carotenoid accumulation in citrus. Overall, our study provides new insights into the molecular regulatory mechanism of carotenoid enhancement in the evolution of citrus, which can facilitate breeding and cultivation efforts to improve the nutritional quality and esthetic value in citrus and hopefully other fruit crops.