AbstractArbuscular mycorrhizal fungi (AMF; Glomeromycota) are difficult to culture; therefore, establishing a robust amplicon-based approach to taxa identification is imperative to describe AMF diversity. Further, due to low and biased sampling of AMF taxa, molecular databases do not represent the breadth of AMF diversity, making database matching approaches suboptimal. Therefore, a full description of AMF diversity requires a tool to determine sequence-based placement in the Glomeromycota clade. Nonetheless, commonly used gene regions, including the SSU and ITS, do not enable reliable phylogenetic placement. Here, we present an improved database and pipeline for the phylogenetic determination of AMF using amplicons from the large subunit (LSU) rRNA gene. We improve our database and backbone tree by including additional outgroup sequences. We also improve an existing bioinformatics pipeline by aligning forward and reverse reads separately, using a universal alignment for all tree building, and implementing a BLAST screening prior to tree building to remove non-homologous sequences. Finally, we present a script to extract AMF belonging to 11 major families as well as an amplicon sequencing variant (ASV) version of our pipeline. We test the utility of the pipeline by testing the placement of known AMF, known non-AMF, and Acaulospora sp. spore sequences. This work represents the most comprehensive database and pipeline for phylogenetic placement of AMF LSU amplicon sequences within the Glomeromycota clade.