The history of permanent human settlement in the high-altitude regions (>3000 m above sea level [masl]) of the Qinghai-Tibetan Plateau (QTP) is important in understanding human adaptation to this cold "Third Pole" region. The Qinghai Lake Basin was an important corridor used by prehistoric humans migrating to the inner QTP. Pastoralism is currently the most important means of sustaining permanent human settlement in the high-altitude regions of the QTP. However, the lack of reliable proxy measures reflecting prehistoric pastoral activities impedes our understanding of how pastoralism developed. The identification of coprophilous fungi in prehistoric cultural deposits may help refine the history of pastoralism. We collected 21 modern domesticated herbivore dung samples and 66 surface soil samples from the Qinghai Lake Basin for fungal spore analyses. We then evaluated how useful such analyses are for identifying grazing activities. Fifty-three samples were also collected from the JXG2 stratigraphic profile (similar to 10.0-0 ka; 3312 masl) for fungal spore analysis. Results indicate that low and stable values of the total concentration of coprophilous fungi were present from similar to 10.0 to similar to 5.5 ka. Concentrations gradually increased from similar to 5.5 to similar to 4.2 ka, significantly increased from similar to 4.2 to similar to 2.6 ka, and then increased dramatically after similar to 2.6 ka. By combining these results with charcoal concentrations (>50 mu m), lithic artifacts, bones and potsherds recovered from the JXG2 site, we infer that early pastoralism in Qinghai Lake Basin appeared between similar to 6.0 to similar to 5.5 ka and gradually intensified throughout the remainder of the Holocene as herding and farming gradually replaced hunting-gathering as the primary subsistence strategies. These results are supported by pollen records, archeological remains and historical records in the northeastern QTP.