We investigated physiological and genetic differentiation between local, metallicolous (M) and non-metallicolous (NM) populations of the pseudometallophyte Biscutella laevigata (Brassicaceae) in the Tatra Mountains of the Carpathian range and in the northern Carpathian foreland. The investigated plants did not hyperaccumulate Pb, Zn or Cd. Presence of EDTA (ethylenediaminetetraacetic acid) in the substrate caused increased metal uptake (Pb, Zn) only in accessions from the M population. The content of mineral nutrients (Fe, Ca, Mg and K) was decreased in accessions from the M population grown in EDTA-enriched substrate. No significant changes in the metal uptake or mineral status were recorded in plants from the NM population. Genetic variability assessed using amplified fragment length polymorphism (AFLP) was equal in the M and NM populations. No signs of a significant decrease in genetic diversity were found in the M population. There was a strong genetic differentiation between the plants from the M and NM populations, which suggests long-term genetic isolation and vicariance between the populations.