BackgroundMycoheterotrophs, acquiring organic carbon and other nutrients from mycorrhizal fungi, have evolved repeatedly with substantial plastid genome (plastome) variations. To date, the fine-scale evolution of mycoheterotrophic plastomes at the intraspecific level is not well-characterized. A few studies have revealed unexpected plastome divergence among species complex members, possibly driven by various biotic/abiotic factors. To illustrate evolutionary mechanisms underlying such divergence, we analyzed plastome features and molecular evolution of 15 plastomes ofNeottia listeroidescomplex from different forest habitats.ResultsThese 15 samples ofNeottia listeroidescomplex split into three clades according to their habitats approximately 6 million years ago: Pine Clade, including ten samples from pine-broadleaf mixed forests, Fir Clade, including four samples from alpine fir forests and Fir-willow Clade with one sample. Compared with those of Pine Clade members, plastomes of Fir Clade members show smaller size and higher substitution rates. Plastome size, substitution rates, loss and retention of plastid-encoded genes are clade-specific. We propose to recognized six species inN. listeroidescomplex and slightly modify the path of plastome degradation.ConclusionsOur results provide insight into the evolutionary dynamics and discrepancy of closely related mycoheterotrophic orchid lineages at a high phylogenetic resolution.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04302-y.