Microsatellite (or simple sequence repeat, SSR) marker is an important tool for molecular research. We isolated and characterized microsatellite markers for half-smooth tongue sole ( hybridizationhed for (AC) and (AG) of half-smooth tongue sole. We detected 883 positive signal from 1 060 random clones by colony hybridization (positive ratio was 83.3%). The majority of positive clones were between 500–1 200 bp in length. We randomly selected 50 clones for sequencing using 33 pairs of primers that were designed with Primer Premier 5.0. Twenty SSRs were polymorphic. A polymorphism analysis of 48 F₁ individuals revealed that 17 SSRs were consistent with Mendelian segregation and could be used to construct the linkage map. Conversely, 3 SSRs deviated from Mendelian segregation (<0.05). Our results suggest that our approach was useful for isolating large numbers of microsatellite markers and construction of a high density genetic linkage map.