Little is known about the variability of response to heavy metal stress within tree species, although it could be a key for a better understanding of tolerance mechanisms and for breeding. The aim of the present study was to characterize the natural variation of response to cadmium (Cd) in Populus nigra L. in order to understand the mechanisms of Cd tolerance. For that, two P. nigra genotypes, originating from contrasting environments in northern (genotype 58-861) and southern (genotype Poli) Italy, were exposed to Cd stress in hydroponics for 3 weeks. The effect of stress was estimated by measuring biomass production, photosynthetic performance and accumulation and translocation of Cd at the end of the experiment. To better understand the mechanisms of Cd tolerance, the expression of some candidate genes involved in the ascorbate–glutathione cycle (ascorbate peroxidase, glutathione reductase, glutathione S-transferase) and in metal sequestration (metallothioneins) was analyzed in leaves. Biomass production and photosynthesis were affected by the treatment in both clones but the southern clone was markedly more tolerant to Cd stress than the other. Nevertheless, the Cd content in leaves was not significantly different between the two clones and was quite low compared to other species. The content of thiols and phytochelatins (PCs), associated with the transcription profile of the glutathione S-transferase gene, indicated relevant differences in the use of the PCs pathway under Cd stress, which could explain the different tolerance to Cd. The northern clone accumulated thiols but down-regulated the GST gene, whereas the southern clone accumulated PCs and up-regulated the GST gene, which can be useful to complex and detoxify Cd. These results suggest that the glutathione pathway is involved in the differential Cd tolerance of the two genotypes. The natural germplasm of P. nigra represents a valuable resource for understanding tolerance to Cd and for selection of plant material for phytoremediation.