The microbial community structure of traditional Koumiss is bound to be different because of the microenvironment and production process of each family. In this paper， using Pacbio SMRT sequencing technology， based on lactic acid bacteria specific primers， the lactic acid bacteria flora of fresh mare milk and Koumiss in Zhaosu County and Akto County， Ili Kazak Region， Xinjiang were explored. The results showed that the alpha diversity of lactic acid bacteria in fresh mare milk was significantly higher(P<0.005) than Koumiss， and the lactic acid bacteria in fresh mare milk were more abundant. Lactobacillus helveticus (35.52%)， Streptococcus salivarius (10.11%)， Lactococcus lactis (9.49%)， Streptococcus thermophilus (5.96%) and Lactobacillus delbrueckii (5.37%) were the main species of lactic acid bacteria in fresh mare milk. The main lactic acid bacteria species in Koumiss were Lactobacillus helveticus (61.77%)， Lactobacillus kefiranofaciens (14.91%) and Lactococcus lactis (9.76%). Staphylococcus saprophyticus(4.45%) and Staphylococcus aureus (2.62%) whose content in fresh mare milk were significantly higher than Koumiss (P<0.05) were pathogenic. In addition， other pathogenic bacteria were detected in fresh mare milk， such as Streptococcus mitis， Streptococcus agalactiae， Streptococcus gallolyticus， Streptococcus pasteurianus and Streptococcus lutetiensis， which stated that fresh mare milk should not be drink directly. And compared with fresh mare milk， Lactobacillus helveticus and Lactobacillus pontis with higher content in Koumiss were significantly positively correlated (r = 0.582， P<0.01). The results of beta diversity stated that fresh mare milk and Koumiss had different lactic acid bacteria flora characteristics， and the dispersion of Koumiss was higher than fresh mare milk， indicating that the quality of traditional Koumiss was uneven. The lactic acid bacteria specific primers used in this article provide better conditions for mining low-abundance lactic acid bacteria， which provides a more adequate theoretical basis for the industrial production of Koumiss.