In this study, 23 genomic microsatellite DNA markers and 17 express sequence tag (EST)-derived microsatellites were developed and characterized using the fast isolation by AFLP of sequences containing repeats (FIASCO) method and data mining from public EST databases of mandarin fish (Siniperca chuatsi). These polymorphic microsatellite markers were then tested for polymorphism in a wild S. chuatsi population. The number of alleles at 23 genomic SSRs varied from 2 to 19 with an average of 8.0 alleles per locus. The average observed and expected heterozygosities were 0.746 and 0.711, respectively. Of 5361 EST sequences examined, 3.9% (209) contain microsatellites, and di-nucleotide repeats are the most abundant (67.0%), followed by tri-nucleotide (29.7%) and tetra-nucleotide repeats (3.3%). The number of alleles at 17 EST-SSRs varied from 2 to 17 with an average of 8.4 alleles per locus. The average observed and expected heterozygosities were 0.789 and 0.685, respectively. No significant difference of loci polymorphism was found between genomic SSRs and EST-SSRs in terms of number of alleles and heterozygosities. Results of cross-species utility indicated that 13 (52.2%) of the genomic-SSRs and 13 (76.5%) of the EST-SSRs were successfully cross-amplified in a related species, the golden mandarin fish (Siniperca scherzeri).