Orchid mycorrhizal fungi (OrMF) play an important role in seed germination and growth of orchids. In this study, the operational taxonomic units (OTUs) of ITS sequences of mycorrhizal fungi in the roots of three Cypripedium species in the same habitat were obtained by culturedependent (peloton isolation) and cultureindependent (clone library) method, to study the phylogenetic relationship and diversity of OrMF. The results showed that: (1) the pelotons distributed in all of the root fragments; 64 OrMF were isolated, including 63 Tulasnellaceae strains and 1 Ceratobasidiacea strain; 7 OTUs were found, and the representative isolate of every OTU formed typical subglobose or ellipsoid monilioid mycelia in chain arrangement. All of the isolated fungi were asexual hyphae and did not produce asexual spores. (2) OrMF obtained from roots of three Cypripedium species by cultureindependent method were affiliated to Tulasnellaceae, Sebacinaceae, Ceratobasidiaceae and Thelephoraceae; The OTUs of Tulasnellaceae had an absolute predominance in type and quantity; It was Cypripedium tibeticum>C. flavum>C. bardolphianum in OTU type and quantity of OrMF acquired by both culturedependent and cultureindependent approach; And the types and quantity of mycorrhizal fungal OTUs obtained by the culturedependent method were less than the cultureindependent one. (3) The phylogenetic analysis of OTUs of Tulasnellaceae showed that the dominant and nondominant OTUs were distributed in three different clades of the phylogenetic tree; This phenomenon that orchids symbiose with many kinds of relatively distant relatives of OrMF may be related to the environmental adaptability of Cypripedium species; There was no significant difference in the OrMF species richness of different Cypripedium species, but there was a differentiation in the community structure.