Fungal endophytes are an important part of the plant microbiome and participate in maintaining the stability of ecosystems. Culture-based methods are frequently considered in studying fungal communities because of their advantages in providing cultures for further experiments and their low cost. In comparison with next-generation sequencing methods, their ability to assess fungal diversity is questionable. Here, we show how using two different incubation temperatures can improve the yield of fungal endophytes by culture-based methods. Diversity, organ preference and spatiotemporal variability of endophytic fungal communities living within Quercus brantii L. were investigated using two different incubation temperatures. To test the effects of using low-temperature incubation on diversity estimations, twigs and leaves of Quercus brantii were incubated under 25 degrees C and 4 degrees C conditions. Isolation rate and Hill's series of diversity were calculated for each sample. Model-based analysis was used to evaluate the effects of (a) incubation temperature, (b) organ type and (c) spatiotemporal variations on fungal isolation rate, fungal diversity and community composition. Our results showed a strong organ specificity and temporal dynamics among endophytic fungal communities, but the location of the forest stand had only a limited effect on fungal community composition. Low-temperature incubation enabled us to improve culture-based methods by discovering the diversity of cold-adapted fungal endophytes and a significantly different part of the endophytic communities that is usually missed by customary culturing temperature. The isolation of cold-adapted endophytes from a semiarid environment opens a new window to further studies on this kind of fungi in such ecosystems.