A set of expressed sequence tag–simple sequence repeat (EST-SSR) loci has been developed for Arabidopsis lyrata ssp. petraea. From 768 root cDNA clones, 126 microsatellites, including di-, tri-, tetra- and pentanucleotide repeat motifs were identified and primers were designed to 24 EST-SSRs. Eleven loci were subsequently screened on 150 individuals sampled from five natural populations, which revealed three to nine alleles per locus (mean 5.36) and expected heterozygosity (HE) estimates ranging from 0.046 to 0.698. Significant deviations from random mating were observed at 10 EST-SSR loci, likely due to inbreeding (global FIS = 0.151) and population structure (global FST = 0.246).