To study the reproductive system of the putative autogamous species Anthyllis vulneraria in Belgian calcareous grasslands, eight polymorphic microsatellite loci were developed using the sequence-tagged microsatellite profiling technique and 35 plants were typed for all loci. No linkage disequilibrium was found between any pair of loci. Over the loci, the number of alleles ranged from two to five. Observed and expected heterozygosities ranged from 0.053 to 0.688 and from 0.053 to 0.631, respectively. The estimated F-IS values (F-IS = 0.031 and 0.122) are not in accordance with a predominantly selfing reproductive system.