PREMISE OF THE STUDY: Viola elatior (Violaceae) is a Eurasian perennial plant species in which French populations are threatened by anthropogenic pressures. Microsatellite primers were developed to investigate its genetic structure and diversity. METHODS AND RESULTS: Eight microsatellite markers were isolated using next-generation sequencing. Loci were amplified and screened for 138 individuals in 17 populations from France. Two of the eight polymorphic loci presented no variability across populations. The total number of alleles per locus varied from two to four. Observed heterozygosity ranged from 0.051 to 1.000. All primers amplified successfully in the closely related species V. pumila. CONCLUSIONS: This set of microsatellites offers a valuable tool for assessing population genetic diversity of the species to improve its conservation and base management efforts. High observed heterozygosity values probably reflect the particular mating system of the species and suggest an important tendency to clonality.