With the development of next generation sequencing technologies, transcriptome level sequence collections are emerging as prominent resources for the discovery of gene-based molecular markers. In this study, we described the isolation and characterization of 46 novel polymorphic microsatellite loci for Siniperca chuatsi and Siniperca scherzeri from the transcriptome of their F-1 interspecies hybrids. Forty-three of these loci were polymorphic in S. chuatsi, and 20 were polymorphic in S. scherzeri. In S. chuatsi, the number of alleles per locus ranged from 2 to 8, and the observed and expected heterozygosities varied from 0.13 to 1.00 and from 0.33 to 0.85, respectively. In S. scherzeri, the number of alleles per locus ranged from 3 to 9, and the observed and expected heterozygosities varied from 0.19 to 1.00 and from 0.28 to 0.88, respectively. We also evaluated the cross-amplification of 46 polymorphic loci in four species of sinipercine fishes: Siniperca kneri, Siniperca undulata, Siniperca obscura, and Coreoperca whiteheadi. The interspecies cross-amplification rate was very high, totaling 94% of the 184 locus/taxon combinations tested. These markers will be a valuable resource for population genetic studies in sinipercine fishes.