Obscure pufferfish (Takifugu obscurus) is an important aquaculture species in China, and sex control breeding is extremely attractive in this species because the testes of male pufferfish are more popular and expensive. However, the sex-specific marker remains unknown and reliable genetic sex identification method has not been established, which has hampered the development of monosexual aquaculture. In this study, the sex-linked amhr2 SNP site of obscure pufferfish was firstly identified by sequencing and high-resolution melting (HRM) analysis. The males exhibit heterozygous genotype (C/G), while females have homozygous genotype (C/C), which is identical to its closely-related species (T. rubripes, T. poecilonotus and T. pardalis). Then, two pairs of male-specific primers were designed based on the sex-specific SNP site and a high-throughput sexing method was subsequently developed with benefits including high specificity and superb sensitivity. Particularly, this method has greatly reduced analysis time and costs compared to the previously established HRM analysis. These results will be beneficial to develop the sex control breeding in obscure pufferfish.