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Development of effective conservation and management strategies requires assessments of ecosystem biodiversity status, especially in understudied hotspots of global fish diversity. Coral reefs are important habitats for fishes, with biodiversity hotspots known globally. We present the first data on molecular diversity of fishes of Mischief Reef, the largest atoll in the Nansha Islands. Partial sequences (650 bp) of mitochondrial COI gene (Cytochrome c oxidase subunit I) are used to identify 209 individuals, representing 101 species, referable to 62 genera, 27 families, 8 orders, and 1 class. The most abundant orders are the Perciformes (176 specimens, 84.21%), Tetraodontiformes (13 specimens, 6.22%), and Beryciformes (13 specimens, 6.22%). Mean Kimura 2-Parameter genetic distances within genera, families, and orders are 4.51, 13.90, and 17.63%, respectively. We record Monotaxis heterodon from this region for the first time—a species that may previously have been misidentified as M. grandoculis. In addition, we recognized possible cryptic species of Lethrinus olivaceus based on significantly diverging barcode sequences. Barcode data provide new insights into fish diversity of Mischief Reef, important for developing further researches on this fauna, and for its conservation.