We assessed the distribution of alien fishes in one of the most ecologically and economically important aquatic ecosystems in Iran, the Karun River Basin. Fish samples were collected from 35 sites in the Karun River Basin during the low-flow period from November to December 2018. We documented the occurrence of 37 species of fishes from nine orders and 14 families. Amongst these, 10 species were alien (986 individuals; 15.7%). The relative abundance of native, endemic, and alien species estimated as 54.8%, 29.5% and 15.7%, respectively. Total length of alien species ranged from 0.4 to 25.5 cm and their total weight from 0.17 to 350 g. The ten alien species belonged to seven families including, Cyprinidae, Cichlidae, Xenocyprididae, Gobiidae, Poeciliidae, Gobionidae, and Salmonidae. Carassius gibelio, Oreochromis aureus, and Coptodon zillii were found to be the most abundant alien species in the downstream region. We applied redundancy analysis (RDA) as a direct stressor method to identify the extent of changes in alien fish assemblages with environmental parameters. The first two axes (RDA1 and RDA2) accounted for 36.24% and 25.33% of the variation of five alien species, respectively. Altitude, depth, electrical conductivity, water temperature, turbidity, dissolved oxygen, and river width were the most significant parameters affecting the distribution of alien species. Our results propose that management practices are needed in the downstream sections of the Basin to recover native and endemic species. Monitoring of presence and extent of alien species is a key to measuring the success of these efforts.