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Abstract Continuous land disturbance could negatively impact microbial community, but perennial crops can potentially reverse this negativity. The objective of this study was to evaluate the effects of Kernza (Thinopyrum intermedium) and alfalfa (Medicago sativaL.) on soil microbial structure and stress condition using the phospholipid fatty acid profiling. The study was conducted at the Ross Jones Research Farm, University of Missouri and consisted of four treatments: Kernza fertilized, Kernza unfertilized, Kernza and alfalfa intercrop, and alfalfa monocrop with four replications. Treatments were established in September 2021 on 18.3 m × 18.3 m plots. Soils from 0‐ to 5‐cm and 5‐ to 15‐cm depths were sampled in September 2021 (before treatments were placed) and 2022 and analyzed for microbial communities. All microbial communities increased after 1 year with the perennial crops. Since differences were not significant among treatments in 2022, this may lead to positive impacts of perennial crops on microbial communities, irrespective of the crop species and management. Moreover, community structure modifications were also observed with the perennial crops, irrespective of the species and management, as evidenced with changes in bacterial community indices in 2022. While fungi/bacteria ratio increased, Gram‐positive/Gram‐negative bacteria ratio decreased in 2022, suggesting a reduction in microbial stress, which can be attributed to ecological functions of the perennial crops. The study showed improvements in soil microbial biomass and modifications in microbial community structure after 1 year of Kernza and alfalfa. As the system matures, relative benefits of management (fertilization and intercropping) and plant species may be realized.