The occurrence and distribution of yeasts in Tibet kefir were investigated in this study. Five samples of Tibetan kefir from Tibet and surrounding areas were collected for yeast isolation. Based on physiological, biochemical characteristics and molecular identification results, eight species of yeast were isolated and identified from Tibet kefir, including Saccharomyces cerevisiae, Pichia fermentans, Debaryomyces hansenii, Rhodotorula mucilaginosa, Candida zeylanoide, Candida parapsilosis, Kluyveromyces marxianus and Kazachstania unispora. Among the test samples, K. marxianus, Ka. Unispora and P. fermentans were the highest three species in frequency of occurrence of yeast isolates. C. zeylanoides, C. parapsilosis and R. mucilaginosa were first found the occurrence in Tibet kefir. The results provided new information of yeast composition and biodiversity of Tibet kefir.