Highland running-water biodiversity has gained growing interest around the world, because of the more pristine conditions and higher sensitivity to environmental changes of highland rivers compared to the lowland rivers. This study presents the findings of systematic investigations and analyses on running-water biodiversity of macroinvertebrate assemblages in the most important headwater streams in the Yalutsangpo and Sanjiangyuan River basins in the Qinghai-Tibet Plateau and the lowland headwaters of the Songhua River, Juma River, and East River during the non-flood seasons of 2012 to 2016. The results indicated that the headwaters on the plateau had a higher regional biodiversity compared to the lowland rivers with the similar flow and substrate conditions. Even though the local diversity of the highland rivers was not significantly different at each single site, the taxonomic composition was significantly different with several rarely seen species scattering among the different sites, resulting in a high regional biodiversity. The biodiversity and composition of macroinvertebrates were strongly affected by the altitude gradient and the environmental variables associated with altitude. To be specific, for the Yalutsangpo River, canonical correspondence analyses of the macroinvertebrate assemblages and their environmental variables indicated that altitude, stream condition (represented by river pattern, riverbed structures, substrate composition), and water temperature influence macroinvertebrate taxa composition. Because of the restrictive plateau conditions including low water temperature, poor aquatic and riparian vegetation, and low runoff, the macroinvertebrate assemblages showed low biodiversity and were vulnerable to potential human disturbance/climate change. Therefore, it is essential to conserve suitable conditions of the determinative environmental variables to protect the unique and high regional biodiversity of the headwaters on the Qinghai-Tibet Plateau.