True flies (Insecta, Diptera) are with number of families one of the most important groups of aquatic insects, which have developed numerous adaptations to the aquatic environment and are found in all aquatic habitats. Larvae and pupae live in aquatic habitats, and with emergence they transfer to terestrial habitats. Adult speciment were collected monthly at the Labudovac and Kozjak-Milanovac tufa barriers in the Plitvice Lakes National Park for 13 years, from February 2007 to the end of 2019. The goal of the study is to determine the diversity and similarity between the composition and structure of aquatic Diptera at both tufa barriers over 13 years with emphasis on the emergence characteristics of the most represented families and preference for microhabitat. 6 emergence traps were installed at the Kozjak-Milanovac tufa barrier, and 6 emergence traps were present at the Labudovac tufa barrier from 2007 to 2011 and 7 emergence traps from 2012 to 2019. The traps differ from each other in the substrate and the water flow rate. The collected material was isolated up to the order, and the order Diptera was identified up to the family level. During 13 years, 79222 individuals from 13 families of aquatic Diptera at the Labudovac tufa barrier and 66785 individuals from 16 families of aquatic Diptera at the Kozjak-Milanovac tufa barrier were collected and identified. At the Labudovac tufa barrier the dominant family is Chironomidae, while at the Kozjak-Milanovac tufa barrier the dominat family is Simuliidae . The peak of emergence at both tufa barriers was recorded in the summer months during higher water temperatures and at lower water discharge. The families Empididae, Muscidae, Psychodidae, and Simuliidae show a positive correlation between abundance and water flow rate and they prefere moss from all present substrates, while other families did not show a statistically significant preference to a particular type of microhabitat or water flow rate.