Victoria (Nymphaeaceae), an annual or perennial aquatic plant genus, contains only two species: V. amazonica (Poepp.) J. C. Sowerby and V. cruziana A. D. Orb. Both species have large floating leaves and variable flower colour. Both Victoria species are night bloomers, which have white petals on the first blooming night that then turn pink or ruby red on the second blooming day. The mechanism of the colour change of Victoria petals during anthesis is still unclear. In this study, flavonoids in Victoria petals of both species were evaluated and quantified by high-performance liquid chromatography with photodiode array detection (HPLC-DAD) and by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) for the first time. In total, 14 flavonoids were detected in Victoria petals, including 4 anthocyanins and 10 flavonols. The flavonoid compositions differed across the two species, resulting in different colours between the inner and outer petals. With increased anthocyanin content across blooming days, the colour of Victoria flowers changed over time. The results of this study will improve understanding of the chemical mechanism of colour formation and lay the foundation for selective colour breeding in Victoria.