Orchids are threatened due of their great ornamental and medicinal values, and seed germination entirely dependent on compatible orchid mycorrhizal fungi (OMF). However, understanding the diversity and specificity of orchid-fungus associations is crucial for understanding ecology of these symbiotic relationships and for orchid conservation purposes. Here, we present preliminary results demonstrating the diversity of OMF isolates obtained from four orchid species, Anacamptis morio, Dactylorrhiza sambucina, Orchis mascula and Platanthera bifolia growing in Czechia, Sweden and Italy. These orchids are known to associate with Ceratobasidiaceae and Tulasnellaceae fungi, showing variation in the specificity of orchidfungus associations. The orchid roots were screened for OMF using culture-dependent (isolation from pelotons) and culture-independent (Next Generation Sequencing of root sections) techniques. The poster presents a comparison of the diversity and specificity of orchid-fungal associations across different geographic regions and orchid taxa obtained by these two methodologies and compares their efficiency for OMF detection. The scientific outcomes can provide deeper insights into the functional implications and evolutionary dynamics of these symbiotic relationships.