Abstract: Arbuscular mycorrhizal fungi (AMF) mediate the interactions between plants and soils, play crucial roles in terrestrial symbiosis, and are important components of soil microbial communities. However, information on the variations of soil AMF communities with respect to the loess soil properties is limited. Therefore, the present study investigated soil AMF diversity, community structure, and physicochemical properties in Medicago sativa fields and farmland in the Loess Plateau semi-arid area. Soil samples (0–20 cm) were collected in June 2021 from four treatments: maize (Zea mays) field (Farmland) and M. sativa fields established in 2019 (L₂₀₁₉), 2012 (L₂₀₁₂), and 2003 (L₂₀₀₃). Illumina MiSeq high-throughput sequencing and real-time fluorescent quantitative PCR were used to explore the structure and diversity of the AMF communities under the four treatments (Farmland, L₂₀₀₃, L_2012, and L₂₀₁₉). Statistical methods (redundancy analysis and molecular ecological network analysis) were used to explore the relationship between soil physicochemical properties and the AMF community. Zero-model analysis was used to reveal the assembly process of the soil AMF community. The results showed that long-term alfalfa planting decreased soil total phosphorus and available phosphorus contents. The AMF gene abundance ranged from 1.02×10⁴ to 1.50×10⁴ copies∙g⁻¹ in dry soil, which was significantly higher in M. sativa field planted in 2003 than in other treatments (P<0.05). Correlation analysis between the abundance of AMF genes and physicochemical factors showed that soil AMF gene abundance was positively correlated with total nitrogen content and negatively correlated with total phosphorus and available phosphorus contents. One class, four orders, seven families, and seven genera of AMF were identified. Glomus, Diversispora, and Paraglomus were the common genera of M. sativa fields and Farmland, and the dominant genera of M. sativa fields and Farmland were Glomus (65.15%−99.12%), mainly contributing to the changes of soil AMF community structure in different treatment groups. Long-term cultivation of M. sativa propagated rare microbial taxa, including Ambispora and Scutellospora, whereas Pacispora and Acaulospora were sterilized. Ambispora was significantly higher in M. sativa field planted in 2019 than in the other treatments (P<0.05). The analysis of the molecular ecological network showed that there were highly abundant genera (Glomus and Paraglomus) that had cooperative relationships in the ecological network, whereas the low-abundance genera (Pacispora and Acaulospora) had competitive relationships in the ecological network. RDA showed no main environmental factors affecting the AMF community structure. The null model was used to infer AMF community assembly processes. In Farmland and M. sativa field established in 2019, community mechanisms were dominantly assembled with deterministic processes (66.67%), with heterogeneous selection contributing the most. For the M. sativa field established in 2012 and 2003, the community mechanisms were dominantly assembled with random processes (100.00%); the undominated processes contributed the most to M. sativa field planted in 2012, and dispersal limitation contributed the most to M. sativa field planted in 2003. The Mantel test showed no main environmental factors driving AMF community assembly. Long-term cultivation of M. sativa increases the number of random processes. This is beneficial for maintaining the sustainability and stability of the artificial grassland ecosystem functions. In summary, long-term M. sativa planting significantly affected the composition of soil AMF communities. This study provides basic data and a theoretical basis for further studies on the microbial mechanisms of AMF on the Loess Plateau after years of M. sativa planting.