Mycoheterotrophic plants rely entirely on fungal symbionts for nutrients, yet the role of intraspecific genomic variation in shaping symbiotic adaptation remains unclear. Gastrodia elata is a mycoheterotrophic orchid with multiple cultivated varieties. Here, we generated a chromosome-level genome of G. elata Bl. f. glauca. Comparative genomic analyses with published G. elata assemblies revealed extensive intraspecific variation, characterized by transposon-mediated inversions occurring in 26% of syntenic regions. Notably, these regions frequently harbored orphan genes. Population genomic analysis of 150 individuals identified three genetically distinct clades: two cultivated (Clades E and G) and one hybrid (Clade I). Transcriptomic profiling uncovered clade-specific expression patterns in symbiosis-related genes, particularly within strigolactone signaling pathways. Molecular dynamics simulations and protein interaction assays demonstrated that polymorphisms in the M domain of the suppressor protein DWARF53 (GeD53) modulate strigolactone signaling by altering the stability of its interaction with the receptor (GeD14). Specifically, a Clade G-specific haplotype enhanced signaling through stabilized protein interactions, thereby influencing tuber development genes, whereas GeD14 variants had minimal functional impact. Further co-expression networks identified LOL5, RNP1, and MTHD as downstream effectors correlating with clade-specific tuber phenotypes and carbohydrate allocation. These findings demonstrate how intraspecific variation in strigolactone signaling components drives functional divergence in G. elata, providing both mechanistic insights into mycoheterotrophic adaptation and genomic resources for future research.

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