Rubber tree [Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg.] is world’s major source of natural rubber and is a native species of the Amazon forest. Although Brazil is the center of origin and genetic diversity of the species, nowadays the country imports around 70% of the rubber it consumes. EST-SSRs (Expressed Sequence Tag-Simple Sequence Repeats) markers, despite showing a lower level of polymorphism than genomic SSRs, are more stable and gene-directed, having the possibility to be involved in the expression of important characteristics. As such, these markers became an important tool in breeding programs and in diversity studies. In this work 20 new developed EST-SSRs markers were characterized in 15 accessions of H. brasiliensis. The maximum number of alleles per locus were 4 and 12 loci were monomorphic. The PIC values ranged from 0.12 to 0.61 and the observed and expected heterozygosity ranged from 0.13 to 0.66 and 0.13 to 0.69 respectively. A cross-amplification of these EST-SSRs loci was tested in six other species of Hevea genus and transferability was observed for 19 markers. Most markers (18) were transferred to species H. guianensis and H. benthamiana, while H. camargoana showed transferability for 16 markers. The polymorphic EST-SSR markers were capable to separate the other species of genus Hevea from H. brasiliensis accessions, creating two groups. These data are important for genetic breeding and can also be used for studies of conservation of species.