Cultivated clones of Hevea brasiliensis have a narrow genetic base. In order to broaden the genetic base, it is first necessary to investigate the genetic diversity of wild populations. Expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed to investigate the genetic diversity of Hevea populations. Four hundred and thirty microsatellites were identified and 148 primers were designed to amplify the loci. Twenty-nine primer pairs were synthesized and evaluated for their ability to detect genetic polymorphisms among 40 wild accessions of H. brasiliensis. Twenty-one of the 29 loci were polymorphic. The number of alleles per locus in the 40 accessions ranged from 2 to 7. HO and HE at each locus ranged from 0.0000 to 0.9000 and from 0.0000 to 0.8704, respectively. All 21 loci could amplify in H. brasiliensis, H. pauciflora, H. nitida, H. spruceana, and H. camargoana. The EST-SSR primers developed herein can be used in genetic diversity and structure studies in H. brasiliensis.