Nitrogen limitation has been well documented in grasslands on the Qinghai-Tibet Plateau (QTP), significantly affecting predictions of plant growth and carbon sequestration potential under future climate change scenario. Beside atmospheric deposition, asymbiotic nitrogen fixation (ANF) may be crucial for nitrogen input in QTP grasslands, due to the lack of artificial fertilization and legume plants. However, little is known about the ANF’s contribution to nitrogen input on the QTP. To fill this knowledge gap, we studied the composition, diversity and activity of ANF diazotrophs across the QTP grasslands by using multiple methods of transect sampling, ¹⁵N-labeling and DNA stable isotope probing (SIP), amplicon sequencing, Random Forest algorithm modelling and digital mapping. We found that Skermanella and Mesorhizobium were the most abundant diazotrophic genera. Soil pH and total phosphorus concentration were the dominant driving factors for their composition and diversity. DNA stable isotope probing with ¹⁵N₂ revealed that Mesorhizobium were the most active nitrogen-fixing microorganisms. The potential N-fixation rates of these diazotrophs ranged from 0 to 18.1 kg N ha^–1 yr^–1, resulting in an estimated annual input of approximately 0.50 Tg N across the entire QTP’s alpine grasslands (i.e., ~25% of annual nitrogen input). The most important factor affecting the ANF rate was soil micronutrient molybdenum, a cofactor in the nitrogen-fixing nitrogenase, accounting for 24.64% of the variance. These findings suggested that ANF diazotrophs play important roles in maintaining nitrogen balance in the QTP grasslands and expand our understanding of Mesorhizobium’s ecological roles beyond traditional symbiotic interactions.