<p>Alfalfa (<i>Medicago sativa</i> L.), known as “Queen of forages”, is valued to its high-nutritional quality and is a key member of Leguminosae family. Its productivity is largely attributed to mutualistic symbioses with arbuscular mycorrhizal fungi (AMF) and rhizobia, which facilitate nutrient exchange and plant growth. However, the coexistence and mutualistic interactions between rhizobia and AMF across alfalfa genotypes with differing yields in native soil remain poorly understood. In this study, we investigated the community composition of rhizobia and AMF colonizing alfalfa roots across different-yield varieties. Our results showed variations in dominant microbial taxa and the structural complexity of root-associated microbial networks among genotypes. Moreover, rhizobia exhibited no significant associations with AMF on genus level, however, negative correlations were observed among genera within the AMF community, and a comparable trend was identified among rhizobial taxa. In summary, our findings offer new insights into how native soil microbiota influence the dual symbiotic relationships of alfalfa, with implications for leveraging native microbial communities to enhance sustainable forage production.</p>