This study was designed to develop and characterize a preliminary set of nuclear microsatellite markers for Aspidosperma polyneuron, an endangered tropical tree species. Leaf samples were collected from a group of mature A. polyneuron trees in a large area along the Pardo and Mogi-Guaçu river basins in São Paulo State in the southeastern region of Brazil. Twenty-five simple sequence repeat (SSR) markers were developed and 16 of 30 samples had polymorphic loci. The number of alleles per locus ranged from two to 17 (average = 7.06). A high diversity (H(e) = 0.65) and parentage exclusion probability for the first (0.9995) and second (0.9999) parents were measured. These 16 polymorphic markers suggest a high potential for genetic diversity, gene flow, and mating system analyses in A. polyneuron.