Chironomidae larvae play an important role in the food chain of river ecosystems in Korea, where it is dominant. However, detailed information on the diet of Chironomidae larvae are still lacking. The purpose of this study was to identify the gut contents of 4th instar larvae of a Chironomidae inhabiting four large-scale weirs (Sejong Weir, Juksan Weir, Gangjeong-Goryeong Weir, and Dalseong Weir) using a DNA meta-barcoding approach. We found that dominant Operational Taxonomic Unit (OUT) was assigned to Paractinolaimus sp. (Nematoda), and the sub-dominant OTU was assigned to Dicrotendipes fumidus (Chironomidae). The most common OTUs among the individuals included phytoplankton, such as Tetrahymena sp., D. armatus, Pseudopediastrum sp., Tetradesmus dimorphus, Biddulphia tridens, and Desmodesmus spp. We calculated the selectivity index (E’) and provided scientific evidence that Chironomidae larvae have a significant preference (E’ > 0.5) for Desmodesmus armatus, E. minima, and T. dimorphus, while it does not show preference for other species found in its gut. Differences in physico-chemical factors, such as water quality, nutrients, Chl-a, and carbon concentrations, resulting from anthropogenic impacts (i.e., construction of large-scale weirs) as well as the particle size of prey organisms (small-sized single cell) and effects of chemicals (chemokinesis) could affect the feeding behavior of Chironomidae larvae.