Roche 454 GS FLX, the second-generation sequencing technique, can read longer fragments accurately and isolate SSR markers, and thus has been widely used in many fields of research. In this study, we applied Roche 454 GS FLX to isolate the microsatellite loci and developed the identification of microsatellites markers in Pleuronectes yokohamae. (AC)12, (AG)12, (AAT)12, (AGG)8, (AGC)8, (AGAT)8 and (ACAG)6 were hybridized with shotgun library of DNA samples. This SSR library was constructed through magnetic beads enrichment, cleaning, elution and purification. The SSR library was then sequenced with Roche 454 GS FLX. MISA was used to search for microsatellite motifs and primer 3 was used to design the primers for P. yokohamae. FAM, HEX, and TAMRA were used to label the microsatellite primers (5′), which were used in the triplex PCR or the nested PCR to identify the genotype of the microsatellite loci. The cluster similarity comparison method was first used to analyze 5641 loci that we obtained, and 247 types of loci with high polymorphism were screened. Among these loci, 52.22% were perfect, 20.24% were imperfect and the rest 27.54% were compound. The percentage of (AC)n and (AG)n was 44% in these loci, and 87.5% of them were more than 10-time repeats. Eleven pairs of fluorescent primers were designed according to the selected loci (more than 10-time repeats) and were applied in 5 individuals. The number of alleles per locus ranged from 3 to 8 with an average value of 5. The average values of Ho, He and PIC were 0.588, 0.788 and 0.670 respectively. Four loci among 11 deviated from Hardy-Weinberg. The results of this research indicated that 454 GS FLX was an intuitive and efficient technique for microsatellite isolating. Our study provides essential information about the population genetic diversity of P. yokohamae, and helps improve the artificial breeding.