Eco-geographical regional system is important for the study of global environmental changes and sustainable development, and it serves as a scientific basis for rationally managing and sustainably utilizing ecosystems and natural resources, such as constructing healthy eco-environments and making policies of environmental management. This paper explained the necessity of validation in the demarcation of eco-geographical regions, which is difficult and may be achieved with some assumptions and presumptions because of the existence of transition zones. Also in this paper, we explored the use of geostatistics in validating regions and boundaries using a case study in Qinghai-Tibet Plateau on the basis of normalized difference vegetation index (NDVI) data. The results show that: (1) eco-geographical regions have different spatial complexity and spatial heterogeneity [i.e. different characteristic values (nugget/sill, fractal) of NDVI], and regions with similar patterns of temperature and moisture have similar mean NDVI values and spatial characteristics [i.e. similar spatial characteristic values (nugget/sill, fractal) of NDVI]. Thus, based on the similarity of spatial heterogeneity or spatial patterns of distribution, demarcations of eco-geographical regions with similar conditions of temperature and moisture, such as IID1 (Ngari montane desert-steppe and desert zone), IID2 (Qaidam montane desert zone), and IID3 (Northern slopes of Kunlun montane desert zone), meet the regional validation requirement. (2) Based on the comparison of spatial heterogeneity or spatial patterns of distribution, the boundary between IIA/B1 (Western Sichuan-eastern Xizang montane coniferous forest zone) and IB1 (GologNagqu high-cold shrub-meadow zone) meets the boundary validation requirement. This boundary guarantees high similarities in an intra-region and high differences in inter-regions, because the value of fractal dimension is the minimum in buffer 1. Furthermore, this paper discussed the application of geostatistics in the choice of index system for boundaries of eco-geographical regions and the determination of region size. The results indicate that the application of geostatistics in eco-geographical regional system is broad, and such researches can serve for obtaining more reasonable and applicable eco-geographical regionalization schemes.