The development of single nucleotide polymorphism (SNP) markers provides the opportunity to improve many areas of plant breeding and population genetics. Unfortunately, for species such as the rubber tree (Hevea brasiliensis), the use of next-generation sequencing for genomic SNP discovery is very difficult because of the large genome size and the abundance of repeated sequences. Access to a set of validated SNP markers is a significant advantage for rubber researchers who wish to apply SNPs in scientific research. Here, we performed genomic sequencing of H. brasiliensis and generated 10,993,648 short reads, which were assembled into 10,071 contigs (N50 = 3078) by a de novo assembly strategy. A total of 2446 contigs presented no hits in the current H. brasiliensis genome assembly and may therefore be considered novel genomic sequences of rubber tree. A total of 143 putative polymorphic positions were selected, gene annotations were available for 58.7 % of the markers, and all of the sequences could be anchored to the released H. brasiliensis genome. These SNPs were validated in eight genotypes of H. brasiliensis and 15 F1 plants from a mapping population, resulting in 30 (20.9 %) positions correctly classified. The analysis revealed key candidate genes responsible for defence mechanisms and provided markers for further genetic improvement of Hevea in breeding programmes. (Résumé d'auteur)