Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.