Summary• The D2 region of the large subunit (LSU) ribosomal RNA gene of isolates of Entrophospora infrequens from trap cultures, the type fungus for the genus Entrophospora , was investigated for sequence variation.• LSU rRNA genes were amplified using PCR from multiple (50 spores) and six single spore DNA extractions. Recombinant clones (261) from these amplifications were analysed for sequence differences using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and sequencing.• Single spores of glomalean fungi have been previously shown to contain high levels of ribosomal RNA gene sequence diversity. From single and multiple spore extractions, 64 glomalean sequences were obtained, of which 61 were unique. These were related to two glomalean families: the Glomaceae (41 / 61) and the Gigasporaceae (20 / 61). No evidence of Acaulosporaceae-like sequences was found.• Sequences related to both families were found within three single spores. Sequences related only to the Gigasporaceae were found in two single spores. The remaining spore only contained sequences related to the Glomaceae. The multiple spore PCR contained sequences related to both families.• The implications of these results for the current taxonomy of this unculturable species are discussed.

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