Ultramafic soils are widespread in the Balkans. Albania and Greece are the richest in the number of endemics, including several hyperaccumulator species, growing on serpentine. The objectives of this study were to understand the potential of Ni hyperaccumulation of these species in close relation with the characteristics of their native soil environments. Collection of both plant samples (analysis of element concentrations in aerial parts) and soil samples (analysis of total elements, DTPA-extractable Ni, Fe, and Ni distribution in mineral phases) allowed evaluation of phenotypic efficacy in hyperaccumulating Ni. Nickel availability in soils is controlled by soil weathering and mineral-bearing phases. Unsurprisingly, the highest levels of Ni availability were associated with amorphous Fe-oxides in moderately weathered Cambisols or with high-exchange capacity clays in well-evolved Vertisols. The highest Ni concentrations in leaves were found in Alyssum murale in Pojska (Albania; 2.0%), Alyssum markgrafii in Gjegjan (Albania; 1.9%), Bornmuellera baldacii subsp. markgrafii in Gramsh (Albania; 1.4%), and Leptoplax emarginata in Trigona (Greece; 1.4%). We identified a new member in the Albanian Ni-hyperaccumulator flora: Thlaspi ochroleucum in Pojska (Albania: 0.13% Ni) and in Pishkash (0.14% Ni). With regard to Ni availability in soils, A. markgrafii (Albania) is the most efficient Ni-hyperaccumulator among all species. Alyssum murale, which is widespread in the serpentines of the Balkans, accumulates Ni, with leaf concentration being negatively correlated to total Ca content of soils regardless of Ni availability (DTPA extractable Ni). If this relationship is confirmed, it would mean that genetic variability is not the main factor that explains the hyperaccumulation performance of this species.