• Premise of the study: Phylogenetic and microsatellite markers were developed for Tulasnella mycorrhizal fungi to investigate fungal species identity and diversity. These markers will be useful in future studies investigating the phylogenetic relationship of the fungal symbionts, specificity of orchid–mycorrhizal associations, and the role of mycorrhizae in orchid speciation within several orchid genera.• Methods and Results: We generated partial genome sequences of two Tulasnella symbionts originating from Chiloglottis and Drakaea orchid species with 454 genome sequencing. Cross‐genus transferability across mycorrhizal symbionts associated with multiple genera of Australian orchids (Arthrochilus, Chiloglottis, Drakaea, and Paracaleana) was found for seven phylogenetic loci. Five loci showed cross‐transferability to Tulasnella from other orchid genera, and two to Sebacina. Furthermore, 11 polymorphic microsatellite loci were developed for Tulasnella from Chiloglottis.• Conclusions: Highly informative markers were obtained, allowing investigation of mycorrhizal diversity of Tulasnellaceae associated with a wide variety of terrestrial orchids in Australia and potentially worldwide.