The natural populations of silver carp (Hypophthalmichthys molitrix) have been declining due to habitat fragmentation, over-fishing and water pollution in China. In this study, 159 transcript-associated microsatellites were developed for silver carp by mining transcriptome sequences, and polymorphism of 27 randomly selected loci were further characterized in a wild population. The number of alleles per locus ranged from 4 to 9, the observed and expected heterozygosity ranged from 0.067 to 0.933 and 0.326 to 0.864, respectively. These novel microsatellite markers will be powerful tools to assess the population structure and genetic diversity in silver carp.