【Objective】The accurate normalisation of quantitative real-time PCR（qRT-PCR）data is contingent on the utilisation of stable reference genes. The objective of this study was to identify and validate suitable reference genes for studying the expression of genes involved in the anthocyanin biosynthesis pathway in two horse chestnut species： Aesculus hippocastanum（common horse chestnut）and A. × carnea（red horse chestnut）are two different species of horse chestnut. Anthocyanins are vital pigments that are responsible for the red，purple and blue colours observed in flowers. In order to comprehend the metabolic regulation of these pigments，reliable gene expression profiling is essential. The selection of optimal reference genes is critical to ensure accurate and reproducible results in qRT-PCR analyses under specific experimental conditions，such as different flower developmental stages and tissue types.【Method】In light of the extant transcriptome data from A. hippocastanum and A. × carnea，a total of twelve candidate reference genes were selected for subsequent validation. The expression stability of the genes under investigation was systematically evaluated across a range of samples，including flowers at different developmental stages and diverse tissues. qRT-PCR was performed to amplify the genes of interest，and their stability was then ranked using four widely recognised algorithms： geNorm，NormFinder，BestKeeper and RefFinder. Each algorithm employs a distinct statistical approach to assess gene stability，thereby providing a comprehensive evaluation. In order to validate the suitability of the selected reference genes，ten key genes involved in anthocyanin synthesis were chosen for expression analysis. These included structural genes such as trans-cinnamate 4-hydroxylase（C4H），chalcone isomerase（CHI），flavonoid 3'-hydroxylase（F3'H），dihydroflavonol 4-reductase（DFR）. The following enzymes and regulatory transcription factors genes were identified in the study： flavonoid 3'5'-hydroxylase（F3'5'H），anthocyanidin synthase（ANS），and flavonoid glucosyltransferase（UFGT），along with regulatory transcription factors genes MYB4，bHLH，and WRKY75. The expression patterns of the genes under investigation were examined in flowers of different colours. The relative expression values were then normalized using the top-ranked reference genes. These values were then compared with transcriptomic data in order to confirm consistency and reliability.【Result】The stability of reference genes varied depending on the sample type. In flowers at different stages of development in both A. hippocastanum and A. × carnea，glyceraldehyde-3-phosphate dehydrogenase（GAPDH），ubiquitin-conjugating enzyme（UBC），and eukaryotic translation initiation factor 5A（EIF5A）exhibited the highest expression stability，while histone H3（HIS3）and polyubiquitin-A（UBQ）showed the lowest stability. In contrast，when different tissues were compared，GAPDH，UBC，and peptidyl-prolyl cis-trans isomerase pin1（CYP）were identified as the most stable，whereas UBQ and tubulin alpha chain（TUB）were the least stable. In general，GAPDH and UBC were found to be the most appropriate reference genes across both sample sets，while UBQ was identified as the least suitable. The validity of these genes was further confirmed by normalising the expression of the ten anthocyanin-related genes using four different strategies. The study utilised a range of experimental groups，including GAPDH alone，UBC alone，a combination of GAPDH and UBC，and a three-gene combination including GAPDH，UBC and CYP. The relative expression levels obtained through these normalisation methods exhibited strong agreement with the expression trends observed in the transcriptome data，thus confirming that the use of these reference genes ensures accurate and reliable quantification of target gene expression.【Conclusion】This study demonstrates that GAPDH and UBC，either as individual reference genes or in combination，are optimal for qRT-PCR analysis of anthocyanin biosynthesis genes in A. hippocastanum and A. × carnea. The stability of these genes across different stages of flower development and various tissue types provides a robust foundation for future expression studies. The utilisation of these reference genes will enable precise investigation into the molecular mechanisms underlying flower colour formation in these species，thereby supporting further functional genomic research and biotechnological applications aimed at modulating pigment accumulation in horse chestnuts.