Members of the non-biting midge family Chironomidae are the most widespread and abundant group of aquatic insects in fresh waters. Although the composition of chironomid communities is considered to be a potential biotic index for water quality assessment, it has not been used easily because of the difficulties with species level identification for larvae. This study was to analyse the molecular operational taxonomic units (MOTU) in chironomid communities to appropriately apply a species level identification. A total of 41 chironomid larvae collected from 7 sampling sites in the Han river system according to water quality were used for analyses. Partial mitochondrial DNA cytochrome c oxidase I (COI) genes were sequenced and analyzed. As a result, 35 different haplotypes (MOTU01-13) were obtained from the 673bp COI sequences. Average genetic distance was 0.0041 (range 0.000 to 0.013) within the MOTU and was 0.1703 (range 0.1181 to 0.2456) between the MOTUs. Nine MOTUs were found at the stream sites represented water quality A, 2 MOTUs at water quality C, and 1 MOTU at water quality D.