Under natural conditions, mycorrhizal symbiosis accompanies nearly the entire life cycle of orchids from seed germination through to foweringfruiting. Tulasnella-like orchid mycorrhizal fungi are the most common mycorrhizal fungi found in association with orchid species. Presently suitable reference genes have not been systematically selected for the quantifcation of gene expression via Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). We evaluated 12 candidate Tulasnella genes in nine diferent Tulasnella isolatesin the Dendrobium-fungal symbiotic germination associations followed by statistical analysis using the programs Bestkeeper, geNorm,Normfnder to analyze the expression stability of the individual genes. The results showed that the EF2, UBC,PP2A genes had the highest rankings with relatively stable expression levels across the diferent genotypesduring the symbiotic seed germination process by the three programs,may be suitable for RT-qPCR normalization. Furthermore, the gene encoding C-5 Sterol desaturase (C5SD) was selected to verify the reliability of EF2, UBC,PP2A expression during the Tulasnella-Dendrobium symbiotic seed germination process. This study is the frst systematic exploration of optimal reference genes for gene expression studies during the colonization of orchid seeds by the mycorrhizal fungus Tulasnella.