Two independently evolved cupricolous populations, their ancestral serpentine population, another unrelated serpentine population and a non-metallicolous population of Silene paradoxa L. were compared for copper tolerance and foliar transcript levels of a 2b-type metallothionein gene that was previously shown to be involved in copper tolerance in the congener, S. vulgaris. The levels of copper tolerance and constitutive MT2b expression, assessed by Northern-blot analysis and semiquantitative RT-PCR, were proportionally increased in the cupricolous populations, as compared to the serpentine and the non-metallicolous populations. Southern-blot analysis revealed amplification of the gene in the cupricolous populations, such as in S. vulgaris, which might account for the higher constitutive expression level. Since the cupricolous populations descended separately from a common serpentine ancestral population, these results imply that MT2b overexpression must have been independently evolved in both of them, again such as previously shown S. vulgaris. Thus, both in S. paradoxa and S. vulgaris, the MT2b locus appears to be a major target for natural selection imposed by soil copper toxicity.