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Plants exhibit remarkable diversity in their petal colors through biosynthesis and the accumulation of various pigments. <i>Lilium</i>, an important cut and potted flower, has many coloring pattern variations, including bicolors and spots. To elucidate the mechanisms regulating spot formation in <i>Lilium leichtlinii</i> var. <i>maximowiczii</i> petals, we used multiple approaches to investigate the changes in petal carotenoids, spot anthocyanins, and gene expression dynamics. This included green petals without spots (D1-Pe and D1-Sp), yellow–green petals with purple spots (D2-Pe and D2-Sp), light-orange petals with dark-purple spots (D3-Pe and D3-Sp), and orange petals with dark-purple spots (D4-Pe and D4-Sp). D3-Pe and D4-Pe contained large amounts of capsanthin and capsorubin and small amounts of zeaxanthin and violaxanthin, which contributed to the orange color. In addition to cyanidin-3-O-glucoside, pelargonidin-3-O-rutinoside, cyanidin-3-O-rutinoside, and peonidin-3-O-rutinoside may also contribute to <i>L. leichtlinii</i> var. <i>maximowiczii</i>‘s petal spot colors. KEGs involved in flavonoid biosyntheses, such as <i>CHS</i>, <i>DFR</i>, and <i>MYB12</i>, were significantly upregulated in D2-Sp and D3-Sp, compared with D1-Sp, as well as in spots, compared with petals. Upregulated anthocyanin concentrations and biosynthesis-related genes promoted spot formation and color transition. Our results provide global insight into pigment accumulation and the regulatory mechanisms underlying spot formation during flower development in <i>L. leichtlinii</i> var. <i>maximowiczii</i>.