Background/Question/Methods: Arbuscular mycorrhizal (AM) fungi form one of the most widespread mutualistic associations with plants, providing nutritional and protective benefits to the hosts in exchange for carbon. One of the biggest challenges to AM ecologists is to characterize the AM fungal communities present inside plant roots. Traditionally, AM fungi are identified based on the morphology of their spores collected from rhizosphere soils. However, spore records are subject to bias. Sporulation rates vary among different AM fungal species, and are highly dependent on biotic and abiotic conditions. Relative abundance of spores is not necessarily indicative of community composition of AM fungi actually infecting plant roots. Recently, a molecular approach—terminal-restriction fragment length polymorphism (T-RFLP) has emerged as an excellent way to characterize AM fungal communities inside plant roots. There is an increasing need of linking T-RFLP profiles to traditional species identifications. The goals of this study were to barcode different AM fungal morphospecies with unique terminal-restriction fragments (T-RFs), and to provide a barcoding database for further investigation of AM fungal communities inside plant roots. Results/Conclusions: Soil samples together with fungal spores and roots were collected from various field locations. In one of the locations—the serpentine grasslands in Chester County, Pennsylvania, spores of ten most common AM fungal species were collected from the soils. DNA was extracted from spores, and partial 18s or ITS regions of RNA genes were PCR amplified using AM1-NS31 primers or ITS4-ITS5 primers, respectively. PCR products were separately digested with different restriction enzymes including HinfI and NlaIII. RFLP patterns were recorded after gel electrophoresis. Our preliminary results showed that while RFLP patterns of the 18s region failed to distinguish some closely related morphospecies, ITS regions were variable enough for assigning unique RFLP patterns to individual AM fungal species. Some of the species such as Glomus etunicatum and G. aggregatum exhibited complex RFLP patterns, indicating these species might possess high intraspecific variation within ITS regions. With one or both primers labeled with fluorescent dyes being used for PCR and the labeled terminal fragments being detected by an automated DNA sequencer, unique T-RFs could be assigned to different species. Therefore, T-RFLP has the potential to barcode different AM fungal species and to provide the means to identification and quantification of these ecologically important fungi residing inside plant roots.