In order to explore the feasibility of highland barley flour freeze-dried powder as fermentation agent to produce highland barley steamed bread, this study used freeze-dried highland barley flour（QKDG） and fresh highland barley flour（QKXX） to produce highland barley dough. Meanwhile, the bacterial colony structure of highland barley dough was compared and analyzed by high-throughput sequencing technology. The results showed that: at the level of bacteroidea, Firmicutes was the dominant population in QKDG and QKXX, accounting for 81.76% and 92.47% of the colony population in each period, respectively. At the bacterial family level, Leuconostocaceae are the dominant family in both QKDG and QKXX, accounting for 70.29% and 48.24% of the community populations, respectively. At the bacterial genus level, Weissella are the dominant genus in both QKDG and QKXX, accounting for 68.41% and 44.39% of the community populations, respectively. From the bacterial community thermogram, we can see that QKDG and QKXX have some differences in the distribution of bacteria, but there is little difference in the microbial community structure between the samples, and there is a certain similarity. Phylogenetic tree showed that the dominant microflora in the QKDG and QKXX samples are Weissella, Lactobacillus, Leuconostoc and Cyanobacteria. The evolutionary relationship between Weissella and Leuconostoc are the closest, followed by Lactobacillus, and Cyanobacteria is the farthest. Through comparisons, it is preliminarily determined that the freeze-dried highland barley flour can be used as the starter for the production of highland barley steamed bread.