Abstract: In 2023, a successful natural breeding experiment for the Acipenser dabryanus was conducted in an artificial simulated spawning environments within natural waters. To assess the effectiveness of this wild breeding experiment and to gain a deeper understanding of the breeding and mating patterns of Acipenser dabryanus, this study employed the mitochondrial D-loop region and 24 microsatellite for parentage assignment and genetic diversity analysis of parents and their offspring. The results showed that among the 20 parental sturgeons, 7 male and 7 female individuals participated in reproduction. Mitochondrial DNA analysis indicated that 240 offspring shared two identical mitochondrial haplotypes with 20 parents. Furthermore, microsatellite loci analysis revealed that these 240 offspring originated from 30 different parental combinations, with 223 offspring successfully matched to their parental lineage, resulting in a high matching success rate of 92.92%. Genetic diversity analysis indicated that the 20 parental sturgeons exhibited a high level of genetic diversity, making them suitable as breeding stock. The genetic diversity of the offspring group was essentially consistent with that of the parents, indicating that the wild breeding experiment effectively maintained the genetic diversity to the offspring group. Three males contributed to 78.92% of offspring, while four females accounted for 91.48% of the offspring. These results demonstrate that A. dabryanus adopts a mixed mating strategy involving multiple females and males. This reproductive strategy is effective for the conservation genetic diversity within the natural population of A. dabryanus. This study not only provides valuable information for the genetic diversity and reproductive genetic management of A. dabryanus but also has significant guiding importance for future wild release and population recovery efforts.