Abstract: To evaluate the fish diversity of national aquatic germplasm resource reserves in Guangdong Province at the molecular level, we collected 550 fish samples from 12 reserves, which were initially identified morphologically as 69 species belonging to 53 genera, 16 families, and 9 orders; by performing DNA barcoding sequencing of the mitochondrial COI gene and combining phylogenetic tree, genetic distance, and haplotype network analyses, a DNA barcode reference database for fishes in the reserves was constructed, with results showing that the species identification success rate of this database reached 98.55% (68/69), confirming its reliability; species delimitation analysis revealed that 7 species including Cyprinus carpio, Cultrichthys erythropterus, and Hemibagrus macropterus formed 2–4 molecular operational taxonomic units (MOTUs) with large genetic distances between units, suggesting the potential existence of cryptic species diversity that may be related to factors such as drainage isolation, complex geological historical activities in South China, artificial release, and misclassification; in contrast, Culter ecurviceps and C. alburnus, as well as Sarotherodon galilaeus and Oreochromis aureus, had small genetic distances and shared one MOTU, showing characteristics of recent divergence. In summary, we established a DNA barcode database for freshwater fishes in national aquatic germplasm resource reserves in Guangdong Province, uncovered cryptic diversity underestimated by morphological identification, and provided an important scientific basis for the subsequent protection and management of fish resources.