In order to study the difference in microbial communities in the highland barley koji from different regions of Tibet， the high throughput sequencing technology was used to analyze microbial community composition and diversity. The results showed that the microorganism groups were rich， and the numbers of bacteria were more than fungus. Bacterial diversity in Naqu (NQ) was the highest， and the fungal diversity in Changdu (CD) was the highest. The dominant bacterial genera were Lactobacillus， Leuconostoc and Weissella in the highland barley koji， the dominant fungal genera were Rhizopus and Aspergillus in the highland barley koji. The microbial community composition in the highland barley koji from different regions of Tibet were certain commonalities and differences. Principal component analysis showed that the bacterial community composition of NQ， Ali (AL)， Rikaze (RKZ) and Shannan (SN) were similar， while that of CD， (Linzhi) LZ and (Lhasa) LS were similar， fungal community of AL， NQ， LS， LZ and SN were clustered into one group， while the CD and RKZ were clustered into one group， respectively. The analysis of the LDA Effect Size showed that there was a bacterial biomarker in all samples， and a fungal biomarker in all samples except for SN sample. In the conclusion， the microbial community composition in the highland barley koji from different regions of Tibet was different. These results could provide theoretical bases for the further development and utilization of probiotic microbial groups and the preparation of mixed jiuqu.