Current ex situ collections of the wild rubber tree Hevea brasiliensis were gathered more than 25 years ago mainly from populations in Southwest Amazonia. Information regarding the structure of genetic diversity among these populations is fragmented and needs refinement. Having analyzed polymorphisms at 15 microsatellites loci throughout the Hevea genome, we report on the genetic diversity of 307 clonally propagated individuals from 19 different collection points. All analyzed loci were highly polymorphic, averaging 21.7 alleles per locus, and displayed heterozygote deficiency. Differentiation among populations was analyzed on a subsample of 220 individuals from 14 populations. The overall F ST is 0.12; although low, this value is significant and suggests moderate differentiation. Mato Grosso (Brazil) populations were genetically more distant from all other populations. A highly significant correlation between genetic and geographical distances over the whole area under study was shown by the Mantel test. The Bayesian algorithm used by STRUCTURE software allowed for the identification of three population clusters that match the boundaries of hydrographical basins of the main Amazon River tributaries. Differentiation among populations could therefore be jointly explained by both geographical location within the hydrographical Amazon network and by isolation by distance among populations belonging to distinct catchments. It is likely that some of these populations no longer exist due to severe deforestation. We provide suggestions to improve genetic resource management and sampling strategy in natural populations of H. brasiliensis.