Ilex paraguariensis A. St.-Hil. (yerba mate) (Aquifoliaceae Bercht. & J. Presl) is a plant species with great economic and cultural importance because its leaves are processed and ground to make infusions like mate or tereré. The species is distributed in a continuous area that includes Southern Brazil and part of Paraguay and Argentina. Uruguay represents the Southern distribution limit of the species, where small populations can be found as part of ravine forests. Although there are previous reports of molecular markers for this and other species in the genus, the available markers were not informative enough to represent the intra- and interpopulation genetic diversity in marginal Uruguayan populations. In this study, we developed highly informative polymorphic microsatellite markers to be used in genetic studies in I. paraguariensis. Markers were identified in contigs from the genome sequence of two individuals and then tested for amplification and polymorphism content in a diverse panel. Markers which passed these filters were tested on populations from Uruguay. They detected higher diversity within populations (in terms of number of alleles and heterozygosity) than previously reported, and levels of heterozygosity similar to those reported for two Brazilian populations. This subset of seven markers were successfully multiplexed, substantially reducing the costs of the analysis. Combined with previously reported nuclear and plastid markers, they can be used to evaluate the genetic diversity of rear-edge populations, identify genotypes for paternity studies and provide relevant information for the conservation and management of germplasm.