Abstract Different primer systems have been developed to characterize arbuscular mycorrhizal fungal (AMF) communities; however, a direct comparison of their specificity, potential to describe diversity and representation of different phylogenetic lineages is lacking. Using seven root samples, we compared four routinely used AMF-specific primer systems for nuclear ribosomal DNA covering i) the partial small subunit (SSU), ii) the partial large subunit (LSU), iii) the partial SSU and internal transcribed spacer (ITS; “Redecker”) and iv) the partial SSU–ITS–partial LSU region (“Krüger”). In addition, a new primer combination v) covering the ITS2 region (ITS2) was included in the comparison. The “Krüger” primers tended to yield the highest AMF diversity and showed a significantly higher Shannon diversity index than the SSU primers. We found a strong bias towards the Glomeraceae in the LSU and SSU primer systems and differences in the composition of AMF communities based on the “Redecker” primer system. Our results confirm the crucial role of the choice of target rRNA marker region for analysing AMF communities. We also provide evidence that nested-PCR based data can be interpreted semi-quantitatively and that the extent of observed AMF community overdominance largely depends on the choice of primer.