• Premise of the study: We developed a set of primers for Encholirium horridum, a species closely associated with inselbergs of the Atlantic Forest, to assess genetic diversity, genetic structure, and gene flow between populations of this species.• Methods and Results: From an enriched genomic library, 10 primer pairs for polymorphic microsatellite regions were developed. The average number of alleles ranged from eight to 20, and the observed and expected heterozygosities ranged from 0.000 to 1.000, and from 0.000 to 0.929, respectively, across the populations.• Conclusions: These markers will be useful in evaluating genetic diversity, spatial genetic structure, analysis of gene flow by paternity, and characterization of mating system of E. horridum.