Venerupis rhomboides is a commercial clam whose production could be enhanced through effective management of natural and hatchery stocks. This study provides the first panel of microsatellite markers for the exploitation of this species according to genetic criteria. A total of 22 polymorphic microsatellite loci were isolated and characterized from two genomic libraries enriched for different motifs. The number of alleles per locus ranged from 2 to 14 in a sample of 20 clams from Spain, and the observed and expected heterozygosity from 0 to 0.95 and 0.05-0.901, respectively. Sixteen loci were in agreement with Hardy-Weinberg equilibrium after sequential Bonferroni correction and linkage disequilibrium between loci pairs was not detected. To reduce the cost of the genotyping process, tri- and pentaplex PCRs, amplifying a total of 13 microsatellites loci were optimized. The microsatellites developed here represent the first nuclear markers described in V. rhomboides and will be useful tools for genetic studies involving assessment of genetic variation and population structure of natural and cultivated populations, assignment testing, construction of genetic linkage maps and dissection of production traits.