Mixotrophic species use both organic and mineral carbon sources. Some mixotrophic plants combine photosynthesis and a nutrition called mycoheterotrophy, where carbon is obtained from fungi forming mycorrhizal symbiosis with their roots. These species can lose photosynthetic abilities and evolve full mycoheterotrophy. Besides morphological changes, the latter transition is associated with a deep alteration of the plastid genome. Photosynthesis-related genes are lost first, followed by housekeeping genes, eventually resulting in a highly reduced genome. Whether relaxation of selective constraints already occurs for the plastid genome of mixotrophic species, which remain photosynthetic, is unclear. This is partly due to the difficulty of comparing plastid genomes of autotrophic, mixotrophic, and mycoheterotrophic species in a narrow phyloge-netic framework. We address this question in the orchid tribe Neottieae, where this large assortment of nutrition types occurs. We sequenced 13 new plastid genomes, including 9 mixotrophic species and covering all 6 Neottieae genera. We investigated selective pressure on plastid genes in each nutrition type and conducted a phylogenetic inference of the group. Surprisingly, photosynthesis-related genes did not experience selection relaxation in mixotrophic species compared with autotrophic relatives. Conversely, we observed evidence for selection intensification for some plastid genes. Photosynthesis is thus still under purifying selection, maybe because of its role in fruit formation and thus reproductive success. Phylogenetic analysis resolved most relationships, but short branches at the base of the tree suggest an evolutionary radiation at the beginning of Neottieae history, which, we hypothesize, may be linked to mixotrophy emergence.