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Spondias tuberosa Arr. Cam belongs to the Anacardiaceae family, an economically important family of plants whose fruits are consumed by humans and animals. The aim of this study was to develop microsatellite markers using sequences from high-throughput sequencing and a magnetic bead enrichment method. The sequences were used to obtain contigs with a minimum of 500 nucleotides using Ray software and the mining of the simple sequence repeats (SSR) was performed with Phobos software, while the primers were designed by Primer3. We developed 18 polymorphic nuclear microsatellite markers and successfully cross-amplified them to three Spondias species. In S. tuberosa, the alleles ranged from 2 to 5 for each locus and Hardy-Weinberg equilibrium was found for 16 loci, with an expected and observed heterozygosity at 0.095-0.755 and 0.1-0.75, respectively. Cross-transferability was obtained for all loci in S. bahiensis, S. dulcis and S. purpurea. We concluded that the microsatellite markers developed in this study are useful in genetic population and conservation studies, as well as for investigating the hybrid origins of Spondias species.