This study investigated the spatial heterogeneity of rhizosphere microbial communities in alfalfa (Medicago sativa) across desertification gradients in Inner Mongolia, China. Rhizosphere soils were collected from non-, lightly-, and moderately- desertified sites. Using 16S rRNA and ITS high-throughput sequencing alongside soil physicochemical analyses, we found that desertification intensity significantly altered microbial structure and function. Actinobacteriota dominated in moderately-desertified soils, whereas Proteobacteria prevailed in non-desertified areas. Ascomycota was the dominant fungal phylum, with Basidiomycota and Mortierellomycota enriched in non- and lightly- desertified sites, respectively. Soil pH and available phosphorus were the key factors shaping bacterial and fungal communities, respectively. Co-occurrence networks indicated enhanced microbial connectivity and a shift toward cooperative interactions under desertification. Functional prediction revealed conserved bacterial metabolic pathways but increased abundance of fungal stress-response enzymes (e.g., monooxygenases). These findings underscore microbial adaptive strategies to desertification and provide insights for sustainable agriculture in arid regions.