As a major component of freshwater ecosystems, insect species play an important role in nutrient cycling and are often used as bioindicators of water pollution. Although extensive studies have characterized insects from freshwater habitats, little is known about the distribution of these species along the Lower Sector of the Danube River. Therefore, this survey conducted in the Danube section within the Romanian territory aimed to identify insect larvae belonging to seven different species of Odonata, Trichoptera, Ephemeroptera, Lepidoptera and Megaloptera by DNA barcoding and to investigate their distribution, density and frequency. A total of 41 quantitative macrozoobenthic samples were collected during two consecutive years (2019 and 2020). Species showed large differences in the distribution and density along different sections, and an overall tendency to populate downstream areas, except for Sialis morio. On the other hand, only Hydropsyche bulgaromanorum, Triaenodes bicolor and S. morio larvae were identified in the upstream section (Sulina branch). These data provide baseline information on the larger range of some of the most common aquatic insects in the Romanian Danube section in relation to several environmental parameters based on the first molecular identification of these species using COI gene sequencing.