The genus Rhinanthus L. is complex, containing many taxonomically unresolved taxa. In this paper we studied genetic variation and species relationships in 15 populations of six Rhinanthus species from three sections. For this purpose, we developed new microsatellite primers for R. osiliensis and used them to investigate genetic variation in two narrow endemics (R. osiliensis, R. javorkae) and in four widespread species (R. rumelicus R. wagneri, R. angustifolius and R. minor). Species-specific private alleles were found in all species except R. osiliensis and R. angustifolius. The Bulgarian endemic R. javorkae showed the lowest genetic variation, followed by widespread R. minor and Estonian endemic R. osiliensis. Rhinanthus javorkae and R. minor were genetically most differentiated. Section Cleistolemus is weakly structured genetically, indicating close affinity between R. osiliensis, R. rumelicus, R. wagneri and R. angustifolius.