Abstract Previous research has found that conventional agricultural systems adversely affect arbuscular mycorrhizal (AM) fungi. However, there is little information on how more ecologically sustainable agricultural practices such as tree-based intercropping (TBI) influence AM fungal communities. In this study, we investigated whether TBI promotes a more abundant and diverse AM fungal community compared to conventional monocropping (CM). Abundance was estimated by measuring spore abundance and hyphal length in soil, and AM fungal colonization of corn (Zea mays) roots. Overall, AM fungal abundance was similar in both systems as corn roots from the CM and TBI systems were heavily colonized (>50%) by AM fungi throughout the growing season. Additionally, soil samples from the CM and TBI systems contained similar spore densities and hyphal length. Molecular analysis of the AM fungal community was assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis of large subunit rRNA genes amplified from roots in the two cropping systems. A total of fourteen AM fungal phylotypes that belonged to the Glomeraceae were found in the two cropping systems. The TBI system had a higher AM fungal richness and contained several taxa not found in the CM system. Molecular analysis of AM fungal communities also revealed significant temporal and compositional differences between the TBI and CM systems. Within the TBI system, tree species differentially influenced the AM fungal community composition in the alley cropping regions. Future research should focus on determining whether compositional differences among AM fungal communities in CM and TBI systems have a functional effect on crop growth and productivity.