To determine an effective DNA preservation method for the detection of arbuscular mycorrhizal (AM) fungi taxa in field-grown sugarcane roots, we compared three low-cost, practical sample preservation techniques—ethanol, silica, and oven drying—with the optimal liquid nitrogen (LN2) technique. In many sugarcane growing regions of the world, access to LN2 is limited or presents logistical challenges. Here, microscopic assessments were undertaken, then DNA extracts were assessed using qPCR assays and DNA metabarcoding before DNA sample preservation methods were evaluated. Samples preserved in LN2 or oven dried yielded optimal results compared with ethanol and silica. Nine AM Operational Taxonomic Units (OTUs) were detected using DNA metabarcoding with four identified to species level. The highest mean total quantities of DNA were obtained using LN2 preservation. However, oven drying was more effective at identifying AM OTUs than LN2 preservation despite yielding lower mean total quantities of AM DNA g−1 root. On this basis, recommendations are made for optimising root sample preservation to obtain AM DNA from sugarcane systems using LN2 and oven drying. As sugarcane is often grown in remote locations or in developing countries where LN2 preservation of roots is impractical, these data demonstrate oven drying to be a viable alternative.