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Premise of the study: To investigate fungal species identity and diversity in mycorrhizal fungi of order Sebacinales, we developed phylogenetic markers. These new markers will enable future studies investigating species delineation and phylogenetic relationships of the fungal symbionts and facilitate investigations into evolutionary interactions among <i>Sebacina</i> species and their orchid hosts. Methods and Results: We generated partial genome sequences for a <i>Sebacina</i> symbiont originating from <i>Caladenia huegelii</i> with 454 genome sequencing and from three symbionts from <i>Eriochilus dilatatus</i> and one from <i>E. pulchellus</i> using Illumina sequencing. Six nuclear and two mitochondrial loci showed high variability (10–31% parsimony informative sites) for Sebacinales mycorrhizal fungi across four genera of Australian orchids (<i>Caladenia</i>, <i>Eriochilus</i>, <i>Elythranthera</i>, and <i>Glossodia</i>). Conclusions: We obtained highly informative DNA markers that will allow investigation of mycorrhizal diversity of Sebacinaceae fungi associated with terrestrial orchids in Australia and worldwide.