Abstract: Acropora corals constitute a key coral group within the coral reef ecosystems of the South China Sea, as they provide structural support to reefs and habitats for numerous marine organisms.Given the diversity and morphological similarity within the Acropora genus, accurate species identification through DNA barcoding is crucial.This study assesses the utility of three genetic markers-cytochrome oxidase I gene (COI), mitochondrial putative control region (mtCR), and nuclear Pax-C intron (Pax-C)-for identifying six morphologically similar Acropora corals in the South China Sea reef ecosystem. Our results showed that all three sequences exhibit significant A+T bias, with Pax-C having the highest mutation rate of 27.3%, followed by mtCR and COI. DNA barcoding gap analysis revealed that the COI gene lacked a barcoding gap. The mtCR sequence showed partial overlap between intraspecies and interspecies genetic distances but still displayed some barcoding gap. In contrast, the Pax-C sequence and the combined mtCR+Pax-C sequences showed a clear barcoding gap. Phylogenetic trees based on the COI gene showed that, except for A. muricata, the other five coral species clustered together. Phylogenetic trees based on mtCR and Pax-C sequences demonstrated that, except for A. hyacinthus, the remaining corals formed distinct clades. Phylogenetic analysis using the combined mtCR+Pax-C sequences showed distinct monophyletic clades for each species. This study confirmed the effectiveness of the combined mtCR+Pax-C sequence as an efficient DNA barcode for identifying closely related six Acropora species, providing scientific support for coral reef ecosystem conservation and management.