In this study, a total of 10 659 Zostera marina EST sequences were downloaded from NCBI database, from which 65 EST-SSR loci were searched with the frequency of 1.7% and the average distributing distance of 21.7 kb. Out of all the repetitive motifs, the trimer was dominant at a ratio of 49.2%, followed by the dimer at a ratio of 33.8%. Based on the searched 65 EST sequences, 30 pairs of primers were designed and synthesized, of which 26 pairs had amplification products with unambiguous bands at similar or larger size than expected. Using eight pairs of polymorphic primers in a population of 16 individuals, the genetic diversity of Z. marina population in Huiquan Bay, Qingdao was analyzed. A total of 33 alleles were obtained at an average of 4.1 alleles per locus, and the genetic diversity of the investigated population was relatively high (Ho=0.538 2; He=0.577 6). The EST-SSR markers developed in this study will provide data for molecular ecology and population genetics study of Z. marina.