We describe nine new variable di- and trinucleotide microsatellites in the plant Crepis sancta, as well as conditions for multiplexing and simultaneous genotyping sets of loci. Their variability was assessed in four populations. Four to 33 alleles were detected per locus; two to 23 alleles per population. Significant deviations from Hardy–Weinberg equilibrium could be attributed either to a mating system involving partial selfing or to null alleles. Genetic differentiation over all loci was significant among the four populations studied. These markers will constitute an efficient tool for investigating dispersal patterns in fragmented landscapes. Cross-species amplification provided few loci variability in three other Crepis species (C. pulcra, C. foetida and C. rubra).