The mitochondrial ribosomal large subunit (Ls) DNA was used to identify the orchid mycorrhizal fungi found in roots of Dactylorhiza majalis. The gene was amplified using DNA extracted from single pelotons obtained from fresh and silica gel dried roots. Furthermore, sequencing a variety of well-characterized orchid isolates expanded the fungal database of the mitochondrial ribosomal LsDNA. Polymerase chain reaction product length variants present in D. majalis were sequenced and identified using the expanded database. These analyses revealed two different peloton-forming fungi in samples from D. majalis, which sometimes occurred together as a single two-taxa peloton within the same cortex cell. The first taxon belonged to the genus Tulasnella and the second taxon was distantly related to Laccaria.