Genetic polymorphism was investigated in Thlaspi caerulescens J. & C. Presl at 15 gene regions, of which seven have been identified to putatively play a role in heavy-metal tolerance or hyperaccumulation. Single nucleotide and length polymorphisms were assessed at four cleaved amplified polymorphic sequences (CAPS) and 11 simple sequence repeat (microsatellite) loci, respectively. The utility of these loci for genetic studies in T. caerulescens was measured among seven natural populations (135 individuals). Fourteen loci rendered polymorphism, and the number of alleles per locus varied from 2 to 5 and 1 to 27 for CAPS and microsatellites, respectively. Up to 12 alleles per locus were detected in a population. The global observed heterozygosity per population varied between 0.01 and 0.31. Additionally, cross-species/genera amplification of loci was investigated on eight other Brassicaceae (five individuals per population). Overall, 70% of the cross-species/genera amplifications were successful, and among them, more than 40% provided intraspecific polymorphisms within a single population. This indicates that such markers may, as well, allow comparative population genetic or mapping studies between and within several Brassicaceae, particularly for genes involved in traits such as heavy-metal tolerance and/or hyperaccumulation.