Rice wine, a traditional fermented alcoholic beverage in China, is produced with grains such as rice, which are fermented with saccharifying starter-koji. Its flavor quality is closely associated to the starter culture-koji, which is made by mixing botanical materials with high-class glutinous rice in certain ecological context. However, there are few reports on the microbial community structure of rice wine koji. In this paper, bacterial community structures of rice wine koji were analyzed using 16S rRNA gene sequencing based on Illumina MiSeq high-throughput technology in 20 samples collected from Xiaogan area, Hubei province and Dazhu area, Sichuan province (10 from each area). We found rice wine koji flora mainly consisted of Weissella, Lactobacillus, Lactococcus, Bacillus, Enterococcus, and Cronobacter, with relative abundances of 29.49%, 10.93%, 8.85%, 4.75%, 1.16% and 1.15%, respectively, as well as an accumulative average relative abundance of 58.71%. They all belonged to Firmicutes and Proteobacteria-the two known dominant genus. Genus-level PCA (Principal component analysis) and OTU-level PCoA (Principal coordinates analysis) based on unweighted UniFrac distances showed that the bacterial community structure differed significantly between the samples from the 2 areas. 7 OTUs were detected in all samples, accounting for 4.4% of the total qualified assembly. Among the 7 OTUs, 3 OTUs were identified as Enterococcus, 2 OTUs were identified as Cronobacter, 1 OTU was identified as Bacillus and 1 OTU was identified as Alkaliphilus. Fifty-eight lactic acid bacteria (LAB) strains were isolated from the 20 koji samples with traditional microbial methods. Among them, Enterococcus faecium and Pediococcus pentosaceus were the dominant LAB isolates, with relative abundances of 51.72% and 31.03%. Despite the differences, a large number of shared bacteria were detected in samples from the two areas.