In hypersaline ecosystems, microbial assemblages are structurally distinctive and play important roles in many microbiological and ecological processes. Here, eukaryotic microorganisms in hypersaline samples were investigated by 454 pyrosequencing of internal transcribed spacer (ITS) gene libraries. In total, 4,645, 1,677, and 5,912 reads were obtained from ITS libraries of waterlogged samples, salt crusts, and saline loess from the alkaline Huama Lake in Shaanxi, China. Analyses of pyrosequencing data revealed that the dominant genera were Dunaliella, Alternaria and Chlamydomonas, which dominated the microbial assemblages in the waterlogged sediments, the salt crusts and the saline loess from the lake banks, respectively. The various infrequent species were not commonly shared by the three types of samples, demonstrating that the eukaryotic microbial compositions of the different environments were distinct. However, the micro-eukaryotic assemblages associated with similar environmental conditions shared some components and were phylogenetically related. The eukaryotic microbial community composition was correlated with the pH value of the site (p = 0.001; r(2) = 0.99), but not with the concentration of total nitrogen or the inorganic ions investigated in this study. The results of this study demonstrated that the hypersaline ecosystems hosted surprisingly diverse eukaryotic microbial community.