A number of recent studies have demonstrated that individual AM fungal species differentially influence soil aggregation and related soil processes, and can have a spectrum of effects on different host plants. Thus, AM fungal species identity and diversity have important implications for agricultural sustainability, both in rangelands, where they may play a central role in determining plant competitive relationships, and in cultivated lands, not least due to their effects on soil aggregation. Due to the generally known limitations of traditional AMF methods for determining AMF community composition actually infecting plant roots, interest in molecular methods has increased in recent years. However, methods based upon AMF ribosomal genes are poorly developed and it isn't readily apparent which methods are optimal for a given application. In this study we examine the utility of the large subunit rRNA gene D1-D2 region for discrimination of AMF species or groups. In addition to sequences containing the FLR3-FLR4 amplicon downloaded from public databases, sequences were cloned from native and weedy plant species of western Montana, aligned and phylogenetic analyses conducted using the neighbor joining algorithm. Phylogenetic group discrimination by terminal restriction fragment length polymorphism (T-RFLP) analyses based on each of eight common restriction enzymes was determined after simulated digestion using the computer program TRFSEQ. These analyses revealed that the FLR3-FLR4 amplicon contains pronounced variability in restriction sites between phylogenetic groups, as well as amplicon length heterogeneity, enabling discrimination of specific groups based on single-enzyme digests. Additionally, the amplicon proved valuable for development of group- and species-specific AMF primers for quantitative analysis purposes. Alignments, T-RF size distributions, primer and probe sequences, as well as current and future results demonstrating field and greenhouse applications are posted on our website (http://biology.umt.edu/fungus/).