Abstract: In order to evaluate the spawning behavior, mating strategies, and genetic contribution of artificially reared Chinese sturgeon population under simulated habitat conditions in 2025, this study employed mitochondrial D-loop sequencing combined with 19 microsatellite loci to perform parentage identification on 25 parental fish and their 75 offspring, and analyzed the parental involvement and pedigree structure. Results indicated that among the 25 parental fish, 2 females (25%) and 4 males (23.5%) successfully participated in reproduction, resulting in the formation of 4 pedigrees; one batch exhibited a “one female–one male” single-pair mating pattern (1162♀ × 1549♂), while another batch followed a “one female–multiple males” hybrid mating pattern. The offspring contribution rate from the core maternal line (1197♀) reached 81.9%, and the success rate of molecular markers in assigning offspring to their parents was 96%. For the 6 parental fish involved in reproduction across the 19 microsatellite loci, the average number of alleles (Na), average observed heterozygosity (Ho), average expected heterozygosity (He), and average polymorphism information content (PIC) were 7.37, 0.740, 0.800, and 0.769, respectively, indicating relatively high overall genetic diversity. Genetic diversity data obtained from the 75 offspring individuals were consistent with those from the 6 parental fish, demonstrating that the offspring population retained the genetic diversity of the parental population. This study confirms that Chinese sturgeons adopt either a “one female–one male” or a “one female–multiple males” hybrid mating strategy during natural reproduction under controlled conditions, providing crucial technical support and theoretical guidance for optimizing habitat restoration techniques for Chinese sturgeons and achieving the self-sustenance of wild populations.