Abstract: Southern Xizang, located within the Himalayan geothermal belt, is one of the most geothermally active regions in China, where microbial eukaryotic communities are under significant environmental pressures. However, research on generalized and specialized microbial eukaryotes in this region remains limited. In this study, we employed 18S rDNA high-throughput sequencing and environmental factor analysis to explore the community assembly mechanisms of generalized and specialized microbial eukaryotes, as well as their responses to environmental changes in 34hot springs in southern Xizang, China. The results showed that the proportion of specialized species was higher, with the dominant phyla including Ascomycota, Chlorophyta, and Arthropoda. Generalized species were mainly composed of Ascomycota, Basidiomycota, and Chordata. The species richness of specialized was significantly higher than that of generalized, although their evenness was lower. β-diversity analysis indicated that both groups were mainly characterized by species turnover. Network analysis revealed that the specialized species exhibited a higher degree of modularity, whereas the generalized species showed stronger connectivity and higher network stability, which may enhance their ability to respond quickly to environmental changes. The community assembly was primarily driven by stochastic processes, with generalized species dominated by undominant processes and heterogeneous selection, while specialized species were more dependent on homogenous dispersal. Among the environmental factors, temperature, latitude, and ammonium nitrogen significantly influenced the distribution of generalized species, with temperature and latitude having a more significant effect. For specialized species, temperature, pH, and altitude were more important, with temperature and pH having the greatest influence. Electrical conductivity may, to some extent, influence the community assembly process of generalized and specialized species. Overall, generalized species exhibited broader adaptability, while specialized species demonstrated stronger adaptability to specific habitats. This study provides new insights into the community assembly mechanisms and ecological functions of plateau geothermal spring ecosystems and offers scientific evidence to global research on microbial diversity in extreme environments.