New microsatellite DNA markers from brown sole were developed and characterized. Fourteen primer sets were designed from 40 microsatellite regions. Eight of 14 loci exhibited variations comprising 8–31 alleles. Observed and expected heterozygosities ranged from 0.611 to 0.833 and from 0.647 to 0.968 among 36 individuals, respectively. Phz3, Phz8 and Phz12 significantly deviated from Hardy–Weinberg equilibrium, and there was a significant linkage disequilibrium between Phz2 and Phz12. Seven of eight loci conformed to the Mendelian manner of inheritance in a full-sib family. Seven to four loci of three related species were cross-amplified by primers for brown sole.