Abstract：Silver carp (Hypophthalmichthys molitrix) is a commercially important fish in China, and its natural resource declined seriously in recent years. Several molecular markers had been used to evaluate population genetics of silver carp, and more markers and samples are still necessary for long term conservation issue. In order to enhance genotypic efficiency of microsatellite DNA(SSR) in silver carp, 14 SSR primer sets with characters of high polymorphism and stable amplification were screened out. Those primers were grouped to three multiplex PCR with four or five primer sets. Parameters of multiplex PCR such as annealing temperature, contents of Taq DNA polymerase，dNTPs，Mg2+ and PCR Buffer were optimized. When the annealing temperature were optimized, the PCR content including 2 units of Taq DNA polymerase，0.4 mmol/L dNTPs，2.3 mmol/L Mg2+ and 3 μL 10×PCR Buffer in a total volume of 25 μL could work preferably for different multiplex PCR. The optimized multiplex PCR then were used to evaluate genetic diversity of silver carp from National Original Breeding Farms in Laojianghe of Jianli and Laohe of Shishou. Two hundred specimens were collected from the two farms and used in this study. Number of alleles ranged from 7 to 24 and from 8 to 20 with mean 13.36 and 12.71 per locus for Jianli and Shishou Farms, respectively. The observed heterozygosity ranged from 0.83 to 1 and from 0.66 to 0.99, with means 0.8479 and 0.9443, and the expected heterozygosity from 0.6462 to 0.9327 and from 0.6075 to 0.9063, with means 0.7967 and 0.8134, respectively. There were 1 and 4 loci being deviations from Hardy-Weinberg equilibrium for Jianli and Shishou Farms, respectively. Analysis of molecular variance (AMOVA) among populations revealed FST=0.0864. It suggested that the genetic diversity level of H.molitrix in both Shishou and Jianli farms were high and no genetic divergence have taken place between H.molitrix populations in the two farms. The results show that the use of those three multiplex PCR can be more rapid and more economic in genetics analysis of H.molitrix
