Pufferfish, commonly known as "fugu," belong to the family Tetraodontidae within the class Actinopterygii. With a wide variety of species, the genus Takifugu has the highest number of members and the greatest economic value. Notable representatives include Takifugu obscurus and T. rubripes. These two pufferfish species are characterized by their tender and delicious flesh as well as their high nutritional value. They are widely used in aquaculture, pufferfish toxin pharmaceutical development, and vertebrate genome research, thus holding significant economic, scientific, and medicinal value. T. obscurus is primarily distributed along the coasts of the Yellow, Bohai, and East China Seas in China. It can also enter the middle and lower reaches of rivers such as the Yangtze River and other connected lakes. It is one of China's important freshwater economic aquaculture species. Due to its high fat content and strong edibility, it is highly favored by domestic consumers, especially those who enjoy cooking. However, issues associated with aquaculture, such as small body size, slow growth, and sensitivity to low temperatures, have led to weaker market competitiveness. T. rubripes is mainly found in the Yellow Sea, East China Sea, and Taiwan waters of China. It is widely cultured in northern China, Japan, and South Korea and is suitable only for marine aquaculture. Compared to T. obscurus, T. rubripes has a faster growth rate and larger body size. However, when facing nutritional deficiency, its larvae may engage in cannibalism. After multiple generations of self-crossing, the germplasm of T. rubripes has degraded, with issues such as deformities and diseases severely impacting breeders. Hybrid breeding combines superior traits of parents to produce new hybrid varieties with improved characteristics. To obtain varieties with better traits and meet the rapidly developing needs of the pufferfish industry, aquaculture farmers have utilized the principle of hybrid vigor to increase production and efficiency. They have crossed T. obscurus (♀) with T. rubripes (♂) to produce hybrid F₁ offspring with good market prospects. The hybrid F1 generation inherits the fast growth rate and large body size of the paternal parent (T. rubripes) and the freshwater culture capability of the maternal parent (T. obscurus). Therefore, the hybrid pufferfish not only retains the freshwater culture capability but also achieved improved growth rates and yields. However, the hybrid F1 generation has morphological features that blended with those of the parents. T. obscurus, T. rubripes, and their hybrid F₁ generations are similar in appearance and body size at the larval, juvenile, and adult stages, making it impossible to distinguish them by naked eye observation. Additionally, certain tissues or processed meat products of pufferfish cannot be differentiated based on appearance alone. In recent years, the hybrid offspring, which share similar morphological features with their parents, have been easily mixed into the parental groups used for hybrid breeding. This has led to prominent issues in pufferfish aquaculture, such as mixed germplasm, serious quality degradation, and uncontrolled hybridization. The escape of hybrid individuals can also affect the gene pool of wild populations, which is not conducive for protecting germplasm resources. Accurate species identification is not only essential for distinguishing pufferfish species but also promotes the rational development of fishery resources, ecological surveys, and biodiversity conservation. Therefore, there is an urgent need for a method to distinguish between T. obscurus, T. rubripes, and their hybrid F₁ generation. In our previous research, we identified a single nucleotide polymorphism (SNP) site in the SH3PX3 nuclear gene, which combined with mitochondrial genes, can be used to identify T. obscurus, T. rubripes, and their hybrid F₁ generation. Direct sequencing of SNP sites can differentiate between the three. However, when faced with a large number of samples, the sequencing cost is relatively high, and the vast and complex data generated during sequencing impose higher demands on researchers' data analysis capabilities. In recent years, the TaqMan probe method based on fluorescence PCR has been widely used for gene expression, mutation, and polymorphism research because of its high sensitivity, speed, and specificity. Compared to ordinary TaqMan probes, TaqMan-MGB probes can accurately distinguish single-base differences and are commonly used for SNP genotyping. In the present study, we designed fluorescence PCR amplification primers and probes based on the polymorphic SNP site of the SH3PX3 nuclear gene, optimized the fluorescence PCR parameters, and established a fluorescence PCR identification method for T. obscurus, T. rubripes, and their hybrid F₁ generation. This method was validated, with the results showing that: (1) The COI sequence of the hybrid F₁ generation was 100% identical to that of the maternal parent T. obscurus, and they clustered together in the NJ phylogenetic tree, making it impossible to distinguish between the hybrid F₁ and the maternal parent; (2) The optimal annealing temperature for the SH3PX3 gene fluorescence PCR system was 48℃; (3) After fluorescence PCR amplification, only the FAM channel of T. obscurus has a Ct value, and the ΔCt is greater than 20, T. rubripes had a Ct value in the FAM channel that was 2 to 5 higher than that in the HEX channel, and the Ct values of the FAM and HEX channels in the hybrid F1 were close, with a difference of less than 2; (4) Based on the above method, 17 samples of T. obscurus, 21 samples of T. rubripes, and 53 samples of hybrid F₁ were verified, with an identification accuracy rate of 100%. The fluorescence PCR identification method established in this study not only provides accurate results and easy interpretation, but also avoids cumbersome processes such as sequencing. It can achieve high-throughput detection and significantly improve detection efficiency. This method offers technical support for the identification and protection of pufferfish germplasm resources, hybrid breeding, and genetic diversity research.