The use of perennial crop species in agricultural systems may increase ecosystem services and sustainability. Because soil microbial communities play a major role in many processes on which ecosystem services and sustainability depend, characterization of soil community structure in novel perennial crop systems is necessary to understand potential shifts in function and crop responses. Here, we characterized soil fungal community composition at two depths (0-10 and 10-30 cm) in replicated, long-term plots containing one of three different cropping systems: a tilled three-crop rotation of annual crops, a novel perennial crop monoculture (Intermediate wheatgrass, which produces the grain Kernza®), and a native prairie reconstruction. The overall fungal community was similar under the perennial monoculture and native vegetation, but both were distinct from those in annual agriculture. The mutualist and saprotrophic community subsets mirrored differences of the overall community, but pathogens were similar among cropping systems. Depth structured overall communities as well as each functional group subset. These results reinforce studies showing strong effects of tillage and sampling depth on soil community structure and suggest plant species diversity may play a weaker role. Similarities in the overall and functional fungal communities between the perennial monoculture and native vegetation suggest Kernza® cropping systems have the potential to mimic reconstructed natural systems.