Neottieae comprise about 150–200 species and are distributed mainly in temperate and subtropical zones of the northern hemisphere. Mycoheterotrophy is common in Neottieae. Based on three DNA markers and a broad sampling of Neottieae, these results indicate that Neottieae is strongly supported as monophyletic and Palmorchis is sister to the remaining genera of Neottieae. Holopogon and Neottia s.s. are deeply nested within Listera. The habit of leafless mycotrophy has independently evolved at least three times in Neottieae, one in Cephalanthera, another in Neottia s.l. and the third in the clade formed by Limodorum and Aphyllorchis.