Background and aims Alyssum section Odontarrhena is the largest single clade of Ni-hyperaccumulator plants, most of which are endemic to ultramafic (serpentine) soils. Alyssum serpyllifolium is a facultative hyperaccumulator able to grow both on limestone-derived and ultramafic soils. Analysis of different populations of this species with contrasting phenotypes could allow the identification of genes involved in Ni-hyperaccumulation and serpentine tolerance.Methods A glasshouse pot experiment on compost-amended ultramafic soil was carried out with three ultramafic (U) and two non-ultramafic (NU) populations of A. serpyllifolium. The leaf ionome was determined by elemental analysis and used as a proxy for serpentine adaptation. A Ni-hyperaccumulating phenotype was estimated from leaf Ni concentrations. Cultured plants were genotyped using Amplified Fragment Length Polymorphism (AFLP) markers. Outlier analysis and regressions of leaf ionome over band distribution were applied to detect markers potentially involved in Ni-hyperaccumulation and serpentine tolerance.Results As well as U populations, some plants from NU populations were found to be able to hyperaccumulate Ni in leaves to concentrations exceeding 0.1% (w/w). U populations had a higher Ca/Mg leaf ratio than NU populations, mainly due to Mg exclusion. 374 AFLP markers were amplified and a potential adaptive value was identified in 34 of those markers.Conclusions Phenotype regression analyses were found to be more powerful than outlier analyses and indicated that regulation of foliar concentrations of Ni, Ca, Mg and P are the main factors involved in serpentine adaptation. More research is needed in order to resolve the ancestral or recently -evolved nature of Ni-hyperaccumulation.< Réduire