Fifteen polymorphic microsatellite loci were developed from an enriched genomic library of the annual plant Rhinanthus angustifolius and characterized using 36 individuals. These markers provided high polymorphism ranging from two to 15 alleles per locus. Four loci showed significant departure from Hardy-Weinberg equilibrium, probably because of the occurrence of null alleles. No significant linkage disequilibrium was detected between pairs of loci. Tests of cross-species transferability were performed on four congeners with a success rate of 100% in Rhinanthus minor, 93% in R. mediterraneus and R. glacialis, and 80% in R. alectorolophus. These microsatellite loci will be useful tools to study mating system, gene flow and hybridization in the genus Rhinanthus.