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Handroanthus heptaphyllus (Vell.) Mattos, popularly known as ipê-roxo, is a species of the family Bignoneaceae much appreciated for its beauty, excellent quality wood which is used for making medicinal products and also in reforestation programs of degraded areas, as well as landscaping and restoration. The aim of this study was to investigate the genetic diversity between and within populations of H. heptaphyllus using microsatellite markers. The 192 seedlings were produced from seeds collected on 30 trees into the two populations of natural forest fragments in Botucatu region, São Paulo, Brazil. Eight microsatellite loci were analyzed, with allelic polymorphism varying from six alleles for locus TAU22 to 14 alleles for loci TAU12, TAU30, and TAU31, with an expected mean number of alleles per locus ( e A ) of 4.9. The mean expected heterozygosity ( e H ) for the two populations was 0.785, the mean observed heterozygosity ( o H ) was 0.609, and the fi xation index (F) was low between populations, with a mean of 0.222. The gene differentiation between the two populations ( ST G ’) was 0.100. We concluded that the higher genetic diversity is within populations; therefore, as far as germplasm collection programs in Botucatu region are concerned, it is recommended that a larger sampling of individuals should be considered within populations, thereby providing good genetic representativeness. The populations have enough genetic diversity to support genetic improvement and germplasm preservation programs.