Mycorrhizal specificity, i.e., the range of fungi allowing mycorrhizal partnerships, differs among orchid species, but that at early developmental stages is unclear. We investigated whether mycorrhizal specificity during seed germination and seedling development differs among three Dendrobium species, D. officinale, D. okinawense and D. moniliforme, in vitro. Nine mycorrhizal fungal strains were obtained from the roots of these species and cultured with a seed of each Dendrobium species. Five to eight fungal strains stimulated seed germination, whereas one to four fungal isolates significantly promoted protocorm development in the three species. To evaluate effects on leafy seedling growth, seedlings obtained from asymbiotic culture were cultured with nine fungal isolates. D. officinale and D. okinawense showed specificity for a single Serendipitaceae or Tulasnellaceae isolate, whereas D. moniliforme exhibited specificity for three isolates of Serendipitaceae and Tulasnellaceae. Therefore, the three Dendrobium species had a growth bottleneck from seed germination to the protocorm stage, and mycorrhizal specificity of protocorm growth and seedling development in vitro varied among the species. Our findings imply divergent mycorrhizal specificity in Dendrobium species at early developmental stages. This study provides insights into the diversity of orchid mycorrhizal specificity, as well as valuable information for conservation of endangered orchids.