Grouper aquaculture constitutes an important industry within marine aquaculture in China. In recent years, inter-specific hybridization has emerged as a prevalent method for grouper breeding, with novel hybrid species, such as the Hulong and Yunlong groupers accounting for more than 70% of domestic grouper production. Although inter-specific hybrid selection has greatly advanced the development of the grouper industry, it has concurrently brought about challenges in species identification and germplasm preservation. In addition, introducing hybrid fish can contaminate the gene pool of species. Hybrid fish possess morphological characteristics of both parents, complicating morphological identification. Therefore, an urgent need exists for precise identification and authentication of hybrid groupers and their parents to ensure the healthy and sustainable development of the grouper industry. This study compared the morphological differences between F1 hybrids and their parents and used the mitochondrial gene CO Ⅰ and nuclear gene RYR3 as dual molecular markers to trace the parents of five common hybrid species: Yunlong, Hulong, Jinhu grouper, Shanhu and Longshu groupers. The results showed the following: (1) The body shape of the hybrid resembled that of the maternal parent, with species, such as Yunlong, Hulong, Jinhu and Shanhu groupers, exhibiting an oval-shaped form, Longshu grouper had a pointed head and humpback. The color and pattern of hybrid groupers combined the characteristics of both parents, among which the Jinhu and Shanhu groupers exhibited high similarity with the color and pattern of their parents, complicating the differentiation between the hybrid offspring from parents based solely on color; conversely, the Yunlong, Hulong, and Longshu groupers exhibited new color patterns through the blending of parental colors. (2) The genetic distance between the five hybrid species and their maternal parents based on the CO Ⅰ gene was 0.000–0.002, all less than 0.020; in their phylogenetic tree, hybrid species were clustered with their maternal parents. The CO Ⅰ gene could accurately identify the maternal parent of hybrid species. (3) Given that nuclear genes transmitted genetic information from both parents, calculating the genetic distance between parental RYR3 genes of hybrids (range: 0.006–0.023) and constructing a phylogenetic tree could effectively distinguish each purebred grouper, indicating that RYR3 was suitable as a molecular marker to assist the CO Ⅰ gene in identifying the parents of hybrid groupers. (4) Based on these results, a method for identifying the parents of hybrid groupers using CO Ⅰ and RYR3 dual molecular markers was established. Initially, the determination of a sample as a hybrid was based on the heterozygosity of its RYR3 gene sequence; subsequent sequence comparison confirmed its parental information; if classified as a hybrid, the parents were identified using CO Ⅰ results. (5) This method was utilized to identify the parents of 16 hybrid grouper samples, and the identification outcomes were consistent with their recorded parental information, demonstrating that the hybrid parent identification method was feasible and effective. These findings indicate that COⅠ and RYR3 dual molecular marker technology can accurately identify the parents of hybrid groupers, providing important insights for the conservation of stingray germplasm resources and genetic breeding.