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Many plant families evolved mycoheterotrophy as a way of gaining nutrients, but the evolution of such nutritional shift has been little studied. We investigated mycorrhizal associations and a nutritional mode of two orchid species, a common ecological generalist Neottia ovata and N. cordata, a species of peatbog spruce forests. The species are closely related to a nongreen N. nidus-avis, associated with ectomycorrhizal Sebacinales, clade A. We investigated the course of germination at 18 sites in the Czech Republic, analyzed fungal spectra of both seedlings and 39 N. ovata and 21 N. cordata adults growing in 32 European sites in different habitats and measured the natural content of stable isotopes. Seeds of both species suffered high mortality during two years of in situ germination. Whereas N. ovata produced few protocorms at both N. ovata and N. cordata localities, N. cordata never reached a protocorm stage. We found Sebacinales, clade B, in both seedlings and adults, independent of habitat type and site geography. The spectra of accompanying fungal species differed according to habitat: the meadow individuals associated frequently with saprophytic Tulasnellaceae, whereas the forest individuals associated more often with ectomycorrhizal fungi. Natural content of stable 13C and 15N isotopes in forest specimens, however, did not indicate significant nutrient gain by mycoheterotrophy. Neottia species form mycorrhiza with Sebacinales, clade B, in various habitats. This association together with the co-occurrence of ectomycorrhizal fungi at forest sites could serve as a predisposition for the shift to full mycoheterotrophy in Neottia. ∑

The genus <i>Neottia</i>, an early step in shift from autotrophy to mycoheterotrophy in orchids?