Isolates of ectomycorrhizal Pisolithus albus were sampled from both ultramafic and volcano-sedimentary soils in New Caledonia, a tropical hotspot of biodiversity, to investigate the relationships between genetic diversity and edaphic constraint through tolerance to nickel (Ni). Carpophore description, spore morphology and phylogenetic analysis based on internal transcribed spacer (ITS) rDNA sequences confirmed that all isolates belong to P. albus and are closely related to other Australasian specimens. Using molecular tools, ITS-restriction fragment length polymorphism and amplified fragment length polymorphism markers, we showed the existence of two distinct genetic clusters within P. albus: ultramafic and volcano-sedimentary. Mycelia response to Ni toxicity supports such a population structure. Pisolithus albus from ultramafic soils included isolates with a high diversity of in vitro Ni tolerance, with both Ni-tolerant isolates (average Ni EC50 at 575 mM) and Ni-sensitive isolates (average Ni EC50 at 37 mM). In contrast, all isolates from volcano-sedimentary soils were found to be Ni sensitive (average Ni EC50 at 32 mM). We highlight that (1) P. albus population from ultramafic soils of New Caledonia are genetically structured in ecotype, and that (2) Ni tolerance among ultramafic isolates suggests an adaptive physiological response to Ni toxicity.