Current studies on the changes in arbuscular mycorrhizal (AM) fungi and root colonization across an altitudinal gradient have mainly focused on alpine environments; however, studies on prairie environments that have a larger area and smaller elevational gradient are markedly insufficient, which limits our understanding of the community composition of AM fungi and their roles across altitudinal gradients. Alpine grassland (mainly composed of cold-resistant and drought-tolerant perennial herbaceous plants), is the largest grassland with the most important ecological functions in the hinterland of the Qinghai-Tibet Plateau. The grassland is characterized by a high altitude (average altitude of 4500-5000 m), with a flat terrain and relatively low elevational change. Therefore, the study of changes in the AM community composition and root colonization across an altitudinal gradient in this extreme environment with low elevational change provides important scientific data for predicting the roles and influences of microorganisms on alpine grasslands as well as understanding the impact of the alpine grassland environment on global environmental change. The present study targeted the alpine grassland in northern Tibet. We analyzed rhizosphere soil samples of colonized alpine grassland plants collected across varying altitudes (4584, 4628, 4744, 4880, and 4956 m) and identified AM fungal spores based on their morphology. The results showed the following: (1) Relatively few genera and species of AM fungi were found in alpine grassland. Four genera, including Acaulospora, Claroideoglomus, Funneliformis, and Glomus, were observed in every altitudinal gradient. The genus Pacispora was not identified in samples collected at 4744 m altitude. The genus Scutellospora was not found at 4744 m or 4956 m altitude, whereas the genus Rhizophagus was only found at 4584 m altitude. (2) The abundance and diversity of AM fungal species significantly decreased as the altitude increased. No significant differences were observed in the Shannon-Weiner index (H) at 4584-4880 m altitude, while a significant decrease in the index was noted at the highest altitude. The numbers and proportions of dominant species showed significant positive correlations with the altitudinal gradient (Funneliformis geosporum and Claroideoglomus claroideum were the dominant species at different altitudes). In alpine grassland, the proliferation and sporulation of AM fungi were sensitive to changes in altitude. Spore density of AM fungi was observed to be distributed in a typical unimodal manner with increasing altitude. Spore density significantly decreased at altitudes below or above 4744-4880 m. (3) The root colonization rate (F, r=-0.779, P < 0.01), intensity of root colonization (M, r=-0.775, P < 0.01), and arbuscular abundance (A, r=-0.556, P < 0.05) of targeted alpine grassland plants significantly decreased with increasing altitude, indicating that the efficiency of root colonization by AM fungi was restricted by the altitudinal gradient. Spore density had no significant effect on F, M, or A. F (r=0.940, P < 0.01), M (r=0.714, P < 0.05), and A (r=0.694, P < 0.05) showed significant increases with increasing altitude. (4) The similarity of the alpine grassland AM fungal community (Sorensen similarity coefficient, 0.821-0.969) was higher at various altitudes, presenting an overall decreasing trend with increasing altitude, which reflects the influence of common species on the AM fungi community. (5) CCA analysis showed that pH, phosphorus, organic carbon, and altitude significantly influenced AM fungal community composition, but compared to the other variables, altitude exerted a higher and more significant impact on the AM fungal community. Thus, changes in soil conditions across an altitudinal gradient play an important role in AM fungal community composition.