Tibetan kefir grains (TKGs), the so-called Tibetan mushroom, contain a variety of microorganisms, e.g., lactic acid bacteria, yeasts, and occasionally acetic acid bacteria, in a white or milky viscous matrix. In the present study, the culturable yeasts in TKGs were isolated, identified and characterized mainly based on scanning electron microscopy, 26S rRNA gene cloning and sequencing, phylogenetic analysis, and fluorescence in situ hybridization (FISH). The results showed most of yeasts in TKGs were spherical (2.0－5.0 μm) or oval (1.0－3.0 × 2.0－4.0 μm). The yeasts were observed present on the outer and inner surfaces of TKGs and were less in numbers compared with bacteria. Totally, sixteen yeast strains were identified, which belonged to three genera-Saccharomyces, Kluyveromyces and Yarrowia, and were grouped to four species of S. cerevisiae (3 strains), Saccharomyces sp. (2 strains), K. marxianus (8 strains) and Y. lipolytica (3 strains).