Novel simple sequence repeats (SSR) were identified from 454 transcriptome pyrosequencing ESTs in the surf clam Mesodesma donacium. From 7,734 ESTs annotated with SSR motives, 75 putative EST-SSRs were selected according their repetitive DNA sequence and functional annotation (Gene ontology), of these, 15 were characterized on 45 individuals of two wild populations located in two different biogeographic transition zones of the coast of Chile (30 and 42A degrees S). Most loci were in Hardy-Weinberg equilibrium, and the polymorphic information content (PIC) ranged from 0.243 to 0.634. Pairwise F (st) between sampled populations was estimated in 0.478. These EST-SSR markers promise to be useful for future management strategies of this overexploited species.