• Premise of the study: Ilex paraguariensis is a native tree species from Brazil, Argentina, and Paraguay that is used in the production of beverages, medicines, and cosmetics. Primers flanking microsatellites were developed to investigate genetic parameters in the species.• Methods and Results: Using microsatellites cloned from an I. paraguariensis shotgun genomic library, 25 pairs of primers were designed and synthesized. Levels of polymorphism were evaluated in 24 individuals from two populations. Twenty loci were polymorphic, and an average of 4.8 and 4.5 alleles per locus were detected in the two populations, respectively. The mean observed heterozygosity was lower than the expected heterozygosity (0.54 vs. 0.60), indicating a departure from Hardy–Weinberg equilibrium and suggesting endogamy in both populations.• Conclusions: The reported set of markers is highly informative and constitutes a powerful tool for the development of genetic characterization studies in I. paraguariensis.