Dairy fan is a traditional milk product made by adding 'acid juice' and heating up. It has been consumed by people of minority ethnic region in Yunnan Province of China for over 1,000 years. In order to improve the quality of dairy fan for further industrial-scale production, we investigated the microbial composition, especially Lactic Acid Bacteria (LAB), by conventional method and 16S rRNA sequences analysis in acid whey, which was the starter culture for traditional dairy fan making. Twenty samples of acid whey were collected from Yunnan. Ninety-one phylotypes of LAB were isolated from these samples and identified by conventional method and 16S rRNA sequencing analysis. The results showed that all isolates belonged to three genera and nine different species. Moreover, Lactobacillus helveticus was the predominant population among these samples, mesophilic and thermophilic LAB could be considered as the major microbial composition of acid whey in the Yunnan Province of China. This paper systematically studied the LAB composition of acid whey, which may be valuable for designing starter culture for dairy fan production.