Abstract In recent years, Illumina MiSeq sequencers replaced pyrosequencing platforms and became dominant in 16S rRNA sequencing. One unique feature of MiSeq technology, compared with Pyrosequencing, is the Paired End (PE) reads, with each read can be sequenced to 250-300 bases to cover multiple variable regions on the 16S rRNA gene. However, the PE reads need to be assembled into a single contig at the beginning of the analysis. Although there are many methods capable of assembling PE reads into contigs, a big portion of PE reads can not be accurately assembled because the poor quality at the 3’ ends of both PE reads in the overlapping region. This causes that many sequences are discarded in the analysis. In this study, we developed a novel approach for clustering and annotation MiSeq-based 16S sequence data, CD-HIT-OTU-MiSeq. This new approach has four distinct novel features. (1) The package can clustering PE reads without joining them into contigs. (2) Users can choose a high quality portion of the PE reads for analysis (e.g. first 200 / 150 bases from forward / reverse reads), according to base quality profile. (3) We implemented a tool that can splice out the target region (e.g. V3-V4) from a full-length 16S reference database into the PE sequences. CD-HIT-OTU-MiSeq can cluster the spliced PE reference database together with samples, so we can derive Operational Taxonomic Units (OTUs) and annotate these OTUs concurrently. (4) Chimeric sequences are effectively identified through de novo approach. The package offers high speed and high accuracy. The software package is freely available as open source package and is distributed along with CD-HIT from http://cd-hit.org . Within the CD-HIT package, CD-HIT-OTU-MiSeq is within the usecase folder.