Flower color, a critical phenotypic trait of ornamental plants, is an essential indicator for flower variety classification. Many physical and internal factors that affect flower color have been widely investigated; however, the effects of internal factors during the flowering period remain unknown. In this study, we evaluated the effects of internal factors on floral coloration during the flowering period of four chrysanthemum cultivars of different colors. colorimetric measurements showed that L*, a*, and b* were in correlation with the lightness and color development in the four chrysanthemum cultivars. The distinctive shape of upper epidermal cells was observed in each flowering stage of different colored chrysanthemums. With progression of the flowering process, the content of anthocyanins and carotenoids increased during early stages, decreased at the senescence stage, and was the highest at the full-bloom stage. The vacuolar pH of flowers gradually decreased as the flower bloomed. Metal contents in flowers varied across different chrysanthemum varieties. Anthocyanins biosynthesis genes, such as CmCHS and CmCHI, were expressed and responsible for pigment changes in red chrysanthemums. Moreover, the expression pattern of cytosol pH-related genes, such as CmVHA-a1, CmVHA-C, and CmVHA-C″1, was in accordance with a decrease in pH during flowering stages. Our results revealed the effects of main internal factors on floral color during the flowering period in four Chrysanthemum varieties, providing insights into the introcellular and molecular regulatory mechanisms of flower coloration and laying key foundations for the improvement of color breeding in chrysanthemums.