Our previous work suggests that Lb.kefiranofaciens is the dominant bacterial species in Tibetan kefir grains and comprises approximately 95% of all lactic acid bacteria. In this research, bacterial isolation and purification, 16S rRNA gene cloning and sequencing, denaturing gradient gel electrophoresis(DGGE), colony PCR and scanning electron microscope observation were performed to understand non-dominant lactic acid bacteria in kefir grains. Scanning electron microscope observation revealed that the cells of Lb. kefiri are short rod (0.8-1.5×0.6-0.7 μm), which were shorter than Lb. kefiranofaciens.Lb. kefiri cells tended to form aggregates in pure culture condition. After sequencing analysis, 1 of 25 clones was related to Lb. kefiri. For 22 colonies isolated from kefir grains, 5 colonies are Lb. kefiri. In addition, DGGE results showed that Lb. kefiri exists in all kefir grain samples. These results demonstrate that Lb. kefiri exists in kefir grain and remains stable and is non-dominant.