Fish diversity is a significant marker of ecosystem health. An updated list of the freshwater fishes of the Euphrates river from the Province of Karbala, middle of Iraq was carried out for the period from November 2019 to March 2021. The freshwater ichthyofauna in Karbala Province comprised 27 species in 11 families and six orders. The most dominant family is Cyprinidae with 12 species, followed by Leuciscidae with four species, and Cichlidae with three species, while eight families contained a single species only. The number of exotic fish species in the Euphrates river, middle of Iraq has increased to eight species (Carassius gibelio, Cyprinus carpio, Hemiculter leucisculus, Heteropneustes fossilis, Gambusia holbrooki, Coptodon zillii, Oreochromis aureus and Oreochromis niloticus). It seems that the impact of the exotic fishes in addition to climatic changes and the construction of dams in the upper parts of the Euphrates river affected the freshwater ichthyofauna in the region.