Microsatellite enriched library of Hyriopsis cumingii was constructed according to the strong affinity between biotin and streptavidin. One hundred clones with foreign inserts were sequenced and 65 clones were screened for usable microsatellites, of which 36 were deemed unique, of sufficient length (more than 8 repeats) and possessed adequate flanking regions for primer designment. Among 36 primer pairs designed, 25 yielded scorable amplification products. Upon testing 30 individuals were sampled using the 25 pair primers from Dongting Lake of Hunan Province, China. Then thirteen polymorphic microsatellite markers were isolated and characterized. These loci exhibited high levels of genetic polymorphism, so the observed number of alleles per locus ranged from 4 to 9. The ranges of observed and expected heterozygosity were 0.2543 to 0.8913 and 0.3629 to 0.8217, respectively, and the average polymorphic information content was 0.5198. Two microsatellite loci were significantly deviated from Hardy-Weinberg equilibrium due to the presence of null alleles, and no linkage disequilibrium found. These microsatellite loci will be useful for assessing the genetic diversity and population structure of H. cumingii.