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Several achlorophyllous orchids associate with ectomycorrhizal hymenomycetes deriving carbon from surrounding trees for the plant. However, this has not been shown for achlorophyllous orchids associating with species of Rhizoctonia, a complex of basal lineages of hymenomycetes that are the most common orchid partners. We analysed Neottia nidus-avis, an achlorophyllous orchid symbiotic with a Rhizoctonia, to identify its symbionts by internal transcribed spacer (ITS) sequencing. Analysis of 61 root systems from 23 French populations showed that N. nidus-avis associates highly specifically with a group of species of Sebacinaceae. Their diversity emphasizes the need for further investigations in the Sebacinaceae systematics. Sebacinoid ITS sequences were often identical within orchid populations and a trend to regional variation in symbionts was observed. Using ITS and intergenic spacer (IGS) polymorphism, we showed that each root system harboured a single species, but that several genets colonized it. However, no polymorphism of these markers was found among portions of each root: this is consistent with the putative mode of entry of the fungus, i.e. from the rhizome into roots but not repeatedly from the soil. In addition, ectomycorrhizae were always found within the N. nidus-avis root systems: 120 of the 144 ectomycorrhizae typed by ITS sequencing were colonized by a sebacinoid fungus identical in ITS sequence to the respective orchid symbiont (even for the IGS polymorphism in some cases). Because sebacinoids were demonstrated recently to be ectomycorrhizal, the orchid is likely to derive its resources from surrounding trees, a mycorrhizal cheating strategy similar to other myco-heterotrophic plants studied to date.