Abstract: Illumina MiSeq is one of the most widely used platforms for human commensal microbiota 16S rRNA gene high throughput sequencing. NovaSeq is the newest Illumina sequencing platform with higher throughput. Although the sequencing principles of MiSeq and NovaSeq are similar, they differ in hardware, software, and reagents, and the similarities and differences in alpha and beta diversity of human commensal microbiota characterized with 16S rRNA gene sequencing at the two platforms remain unclear. In this study, the 16S rRNA gene V3-V4 regions of 10 human fecal samples and 8 human vaginal swab samples were amplified and library was constructed and sequenced at MiSeq and NovaSeq platform, respectively, and identical bioinformatic pipeline was used to process the sequencing data generated by the two platforms. At the same sequencing depth, NovaSeq detected more bacteria taxa of low abundance than MiSeq. At the Phylum, Class, Order, Family, Genus, and amplicon sequence variant（ASV） levels, MiSeq and NovaSeq detected same dominant bacterial taxa, and their relative abundances were correlated significantly, and positively. The abundances of some dominant bacteria in the same sample differed significantly between the two platforms. Alpha diversity indexes at two platforms, including index, Pielou′s evenness, and number of observed ASVs for all samples, and Faith′s PD for fecal samples were significantly, strongly, and positively correlated, the absolute values of the first three alpha diversity indexes significantly differed between the two platforms. At the aspect of beta diversity, the overall microbiota structure of all samples showed no significant difference between the two platforms, and the majority of the differential bacteria ASV between the fecal and vaginal microbiota identified by the two platforms（19 out of 23 ASV） were identical. In conclusion, for human fecal samples and vaginal swab samples, the MiSeq and NovaSeq platforms do not change the relative differences in dominant bacterial abundances, alpha diversity indexes, and beta diversity among varied samples, but indivi-dual samples differ in the numbers and abundances of detected bacterial taxa, and absolute values of alpha diversity indexes between the two sequencing platforms.