Background and aims <p>Nickel is economically important, but its mining is energy-intensive and environmentally damaging. Agromining, which employs hyperaccumulator plants, represents a sustainable alternative. <i>Odontarrhena chalcidica</i> (Brassicaceae) shows a Ni uptake potential promising for agromining. This study aimed at assessing morphological and physiological traits and Ni content of four <i>O. chalcidica</i> genotypes from Northern Greece. For the first time, a multidimensional approach was used to compare plant traits and Ni accumulation of different genotypes across various cultivation systems.</p> Methods <p>Genotypes were phenotyped for morphological and physiological traits. Nickel accumulation in roots, stems, and leaves was quantified. Plants were grown in aeroponics, hydroponics and pot systems to assess trait consistency. Morphological traits were evaluated through biometric measurements and Environmental Scanning Electron Microscopy. Physiological photosynthetic parameters were analysed and chlorophyll content determined. Nickel content was quantified using monochromatic X-Ray Fluorescence spectroscopy.</p> Results <p>Significant phenotypic variability was observed among genotypes. Morphological parameters, such as stomatal and trichome densities, showed stability across cultivation conditions. Hydroponics and aeroponics proved to be suitable for phenotyping, as they did not significantly alter key morphological and physiological traits observed in pot. However, biomass yield and Ni uptake varied across cultivation system for both roots and leaves, with hydroponics being optimal for growth and Ni accumulation.</p> Conclusions <p>The phenotypic diversity observed among genotypes proved to remain consistent across cultivation systems. <i>Odontarrhena chalcidica</i> traits observed in hydroponics and aeroponics are correlated with those observed in pot, thus could be the basis for future field agromining applications.</p>