The influence of agricultural practice on the diversity of Arbuscular mycorrhizal fungi (AMF, Glomeromycota) continues to be an important research question as these mutualistic symbionts are known to improve plant growth and soil quality. To this day, studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, such as the small and large subunits and the ITS region. In the Glomeromycota, these regions show high intra-organism polymorphism, which can seriously complicate interpretation of diversity data. We chose the rpb1 gene as an alternative marker as this putative single-copy gene has been shown to be monomorphic within fungal isolates but shows an excellent barcode gap between species. It has also been widely used for phylogenetic studies in other fungal groups. We designed a glomeromycotan-specific PCR primer set for 454 sequencing of a region of this gene, and established a new reference dataset comprising all major AMF lineages. Using this novel molecular tool, we analyzed the influence of tillage regimes on AMF root communities in a field experiment in Switzerland. The PCR primers showed a high specificity for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and for increased patchiness among non-tilled plots. Tillage effects on different AMF taxa are more complex than previously thought and provide indications about their ability to adapt to agricultural management practices and their life history strategies. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.