The Xinjiang-Tibet Road (G219), located in the western part of the Tibetan Plateau, is an important transportation route connecting Xinjiang and Tibet. There are numerous mountains along the route, including the Himalayas, Gangdisê, Karakorum and Western Kunlun, where glacial lakes are widely developed and the road is susceptible to potential glacial lake outburst floods (GLOFs). Based on Landsat ETM+ and Sentinel-2 MSI images, a combination of the normalized difference water index method and manual visual revision was employed to extract boundaries of glacial lakes within the basins where the Xinjiang-Tibet Road passes in 2000 and 2022. Furthermore, we analyzed the spatial and temporal evolution characteristics of glacial lakes in this region and assessed their GLOF hazards as well as potential affected road sections. The results showed that: (1) In 2022, there were a total of 1412 glacial lakes covering an area 141.13 km² within these basins. From 2000 to 2022, both the number and area of glacial lakes with an area of ≥0.01 km² showed an increasing trend, with an increase of 98 (7.46%) and 2.16 km² (1.56%), respectively. The number and area of glacial lakes below an altitude of 4500 m remained relatively stable, while those above an altitude of 5000 m experienced rapid growth. (2) The GLOF hazard assessment of 70 moraine-dammed glacial lakes with an area of ＞0.1 km² identified 13 very high, 24 high, 13 medium and 20 low cases. Moreover, ten sections along the Xinjiang-Tibet Road that were found susceptible to hazardous GLOFs, especially R1 (K514+210-K514+770), R2 (K793+190-K802+250), R4 (K1286+690-K1287+880), R8 (K1684+900-K1685+380) and R9 (K1786+110-K1801+700). The strengthening of glacial lake dynamic monitoring in upstream of these sections and implementation of corresponding control measures are highly recommended.