The spotted scat, Scatophagus argus, has become a popular commercial fish in recent years. However, spotted scat fingerlings originate mainly from wild resources due to difficulties in the artificial propagation of this species. Thus, an understanding of its population genetic structure is necessary for its conservation management and commerical development. A valuable technique for conservation and evolutionary studies is the examination of single nucleotide polymorphisms (SNPs) using next-generation sequencing. In the present study, novel SNPs were discovered using high-throughput 454 pyrosequencing in the spotted scat (S. argus). The SNPs were detected in 4,825 contigs after de novo assembly. Among them, 33 SNPs were validated with high-resolution melting analysis using 36 individuals originating from 2 wild populations (Dinghai, Zhejiang: latitude 30°0′20″N, longitude 122°06′23″E; Zhanjiang Guangdong: latitude 21°11′43″N, longitude 110°34′56″E). The number of alleles ranged from 2 to 4, and values of the inbreeding coefficient index (F is ) varied from −0.139 to 0.526. Except for Sa_SNP6 and Sa_SNP10, no significant deviation from the Hardy–Weinberg equilibrium was observed. These novel SNPs will contribute to research design and management approaches for the conservation of S. argus.