Background and Aims: An understanding of mycorrhizal variation, orchid seed germination temperature and the effect of co-occurring plant species could be critical for optimising conservation translocation of endangered plants with specialised mycorrhizal associations. Methods: Focussing on the orchid Thelymitra epipactoides we isolated mycorrhizal fungi from ten plants within each of three sites; Shallow Sands Woodland (SSW), Damp Heathland (DH) and Coastal Heathland Scrub (CHS). Twenty-seven fungal isolates were tested for symbiotic germination under three temperature ranges: 12-16°C, 16-24°C or 27°C. Fungi were sequenced using the ITS, nLSU, 28S and mitochondrial regions. Orchids were grown to maturity and co-planted with each of ten associated plant species with tuber width measured at 12 months after co-planting. Key Results: Two Tulasnella fungal lineages were isolated and identified by phylogenetic analyses, OTU1 and “T. asymmetrica”. Fungal lineages were specific to sites and did not co-occur. OTU1 (from the SSW site), germinated seed predominately at 12-16°C (typical of autumn-winter temperature) whereas “T. asymmetrica” (from the DH and CHS sites), germinated seed across all three temperature ranges. There was no difference in the growth of adult orchids germinated with different OTUs. There was a significant reduction in tuber size of T. epipactoides when co-planted with six of the commonly co-occurring plant species. Conclusions: We found orchid fungal lineages and their germination temperature can change with habitat and established that translocation sites can be optimised with knowledge of co-occurring plant interactions. For conservation translocations, particularly under a changing climate, we recommend plants should be grown with mycorrhizal fungi tailored to the recipient site.