<p>Arbuscular mycorrhizal fungi (AMF) are central components of terrestrial ecosystems and agroecosystems. However, their accurate identification remains methodologically challenging due to their complex biology and the limitations of traditional morphological approaches. Over the past three decades, molecular tools have profoundly reshaped AMF research, shifting from spore-based identification and Sanger sequencing of ribosomal markers toward high-throughput amplicon sequencing and, more recently, metagenomic frameworks that enable community-level and functional analyses. This review critically examines the conceptual and technical evolution of AMF identification strategies, comparing morphological characterization, ribosomal DNA markers (SSU, ITS, LSU), multilocus approaches, metabarcoding, and whole-genome metagenomics. We analyze their taxonomic coverage, resolution, and methodological biases, including primer specificity, intragenomic rDNA variation, database limitations, and bioinformatic pipeline effects. Attention is given to how marker selection influences ecological interpretation, cross-study comparability, and functional inference. Finally, we propose practical guidelines for aligning marker choice with study objectives and outline validation strategies—such as mock communities, curated reference databases, and multi-marker integration—to improve reproducibility and taxonomic robustness. By integrating historical perspective, methodological evaluation, and applied recommendations, this review provides a decision-oriented framework to support more accurate and comparable assessments of Glomeromycota diversity.</p>