Abstract: To establish a cost-effective and accurate paternity assignment system for grass carp (Ctenopharyngodon idella) and to assist the research on family selection and breeding, we selected 41 individuals exhibiting traits such as rapid growth and a slender body shape as parent fish and established 22 families. Then we randomly selected 115 offsprings, and conducted parentage identification for them using five highly polymorphic microsatellite markers. The results show that a total of 68 alleles were detected, with an average number of alleles (N_a) per locus of 13.6. The average expected heterozygosity (H_e) and observed heterozygosity (H_o) were 0.825 8 and 0.875 2, respectively. The polymorphic information content (PIC) ranged from 0.527 to 0.906 (average 0.804 4), indicating high polymorphism (PIC>0.5). When both parental sexes were known and the confidence level was set at 95%, the cumulative exclusion probability (CE-PP) reached 99.99%. All 115 offsprings were accurately assigned to their respective parents, achieving an identification accuracy of 100%. This study successfully determines the parental origin of offspring under polyculture conditions, providing a reliable foundation for future selective breeding in C. idella.