Sterility of hybrids produced from interspecific hybridization in lilies (Lilium, Liliaceae) is a great limitation in the breeding program, especially for Lilium longiflorum, which only has white-flowered cultivars. Because modification of flower colour in L. longiflorum by conventional breeding is limited by pre- and post-fertilization barriers, we think genetic modification could be used as an alternative in the future. For this, we need to understand what determines white colouration in L. longiflorum and other species and identify the molecular mechanisms regulating flower colour. In this study, we determined the accumulation of anthocyanins and related compounds in flower tissues during flower developmental stages in L. longiflorum cultivar ‘Lincoln’ and in the Oriental hybrid lily cultivars ‘Rialto’, ‘Perth’ and ‘Gran Tourismo’, respectively with white, pink and red flowers. Furthermore, the presence/absence and the expression of eight structural genes (CHSa, CHSb, CHIa, CHIb, F3H, F3’H, DFR, ANS) and three transcription factor genes (MYB12, MYB15, bHLH2) in flower tissues were investigated. Two structural genes (LlLinF3’H and LlLinDFR) and one transcription factor gene (LlLinbHLH2) were not detected in ‘Lincoln’ flowers. In ‘Rialto’, an amino acid substitution in the R2 repeat of LhRiaMYB12 which was previously reported to be responsible for the white flower colour is also found in the LhPerMYB12 of the pink ‘Perth’ flowers. Moreover, LhRiaDFR is present but not expressed in ‘Rialto’ flowers. Accumulation of cyanidin was observed in the flowers of ‘Perth’ and ‘Gran Tourismo’. High amounts of dihydrokaempferol accumulated in flowers of all four lily cultivars confirming the expression and functionality of early structural genes in the pathway. The elevated expression of the structural genes is strongly correlated with the expression of LhMYB12 and LhMYB15. This information can be used in the future to generate new L. longiflorum or Oriental lily hybrid cultivars with novel flower colours.