Most large rivers in South Korea run through major cities, which often experience many environmental problems, including outbreaks of non-biting midges (Diptera: Chironomidae). However, chironomid species inhabiting large rivers have not been thoroughly investigated. We aimed to identify chironomid species collected from the four main large rivers in South Korea, construct a corresponding DNA barcode library, and examine the distribution and community structure of the identified riverine species. Adult chironomids were collected from nine sites along the rivers by using sweep nets and light traps during June and August 2015. Adults were morphologically identified, and COI nucleotide sequences were generated to verify the species identification and construct a DNA barcode library. The distribution and community structure of the identified species were also analyzed. A total of 124 COI sequences were established from 37 species belonging to 19 genera, and the resulting DNA barcode library effectively discriminated >90% of riverine Chironomidae in South Korea. Ten species, which are considered indicator species for large rivers, were collected from all four rivers. In addition, members of the subfamily Chironominae were collected more frequently than members of other subfamilies, with Tanytarsus tamagotoi being the most common and widespread chironomid species in South Korea. The DNA barcode library developed in this study will facilitate environmental studies of large rivers, such as biomonitoring chironomid larvae.

Chironomid larvae are among the dominant benthic macroinvertebrates in all types of water systems in South Korea. They may pass through pipes in rivers (raw water) and occur in drinking water, thus creating public health issues. However, little is known about the larval stages of chironomids in large South Korean rivers. Therefore, we examined larval–adult associations in chironomids inhabiting major rivers used as water sources. The larvae were collected in 2015 and 2016 from nine locations along the four largest rivers in South Korea using a Ponar grab. Cytochrome oxidase subunit I (COI) sequences were generated from the larval specimens, and the species were identified by comparing these sequences to those in a newly constructed DNA barcode library of Chironomidae in South Korea. The samples from the four rivers yielded 61 mitochondrial COI sequences belonging to 18 species, including Hydrobaenus kondoi Saether, 1989, which was reported for the first time in the Korean Peninsula. Further, morphological identification of the larvae was conducted, and a pictorial taxonomic key to Chironomidae species in large rivers in South Korea was developed to facilitate freshwater biomonitoring research. Finally, an action flow chart was created for the rapid identification of chironomid larvae in infested drinking water or water purification facilities.