In this study, three multiplex PCR panels were established using microsatellite markers developed from our laboratory by optimizing annealing temperature, concentration of primers and times of cycle.A breeding group "JD-01" was analyzed using the three multiplex PCR panels.The results showed:the average number of alleles and effective number of alleles were 12.111 and 7.408 respectively.The average of polymorphism information content(PIC) were 0.846.The average of observed heterozygosity(H_o) and expected heterozygosity(H_e) was 0.825 and 0.868, respectively.The Shannon's diversity index was 2.165.By using Cervus 3.0 software, we verified parentage assignment that the pedigree relationship was known in 9 families of large yellow croaker.As a result, the accuracy rate of parentage assignment was 100% by using three multiple PCR panels.The development of microsatellite multiple PCR system provides a high efficiency technical method for genetic diversity analysis, parentage assignment and marker-assisted family management in large yellow croaker.