An understanding of the genetic diversity and structure of plant species is essential in order to comprehend the degree of biodiversity loss and to develop successful restoration programs. Handroanthus is an important genus that presents one of the most valuable timbers of South America. Handroanthus chrysanthus is an important species distributed in Central and South America. Microsatellite markers are not previously developed for this species. Ten microsatellites for Handroanthus chrysanthus developed using high-throughput sequencing are presented here. The usefulness of these microsatellite loci for the genetic analysis of subspecies H. chrysanthus subsp. chrysanthus (distributed in coastal dry forests) and subspecies H. chrysanthus subsp. meridionalis (distributed in premontane moist forests) is analyzed. At least eight polymorphic microsatellites are useful for each subspecies, seven of which can be used in both subspecies.