Investigations on host affiliations, seasonal variation and physiological response to drought tolerance of dark septate endophytes (DSE) in extremely arid desert environments can help explain the adaptive mechanisms of these endophytes in desert ecosystems. The colonization and species diversity of DSE and the soil factors in the rhizospheres of five desert shrubs were therefore determined in Anxi extremely arid environment in Northwest China in July, September and December 2020. Subsequently, six DSE strains were selected to test the growth and metabolic response of DSE strains to drought stress in vitro. The sampled plants and seasons significantly influenced the morphology, colonization and species diversity of DSE. Of the 15 DSE species isolated in the roots of these plants, Triangularia allahabadensis was reported in desert ecosystems for the first time. DSE colonization was predominately affected by season and soil factors, while species composition was more corresponded to plant species. The composition of metabolites of DSE strains varied significantly due to levels of drought stress and DSE identity, which indirectly reflected the difference of their traits to respond to drought stress. This study confirmed the establishment of relationship between DSE and roots of xerophytic plants, and proved the drought tolerance of DSE in vitro, providing insight into the application of DSE to assist desert plants to resist drought.