This investigation and critical revision of the ichthyofauna of Tajikistan is the result of two nationwide surveys completed by the authors in 2017 and 2019 combined with an analytical examination of previous studies. The ichthyofauna of Tajikistan includes 60 species, of which 44 are native and 15 are nonnative, while one species (Cyprinus carpio) is represented by populations of native and alien origin. Seven species recorded in neighboring countries could potentially be found in Tajikistan, 12 introduced species are nonnaturalized, two were erroneously identified as belonging to the ichthyofauna of Tajikistan, and two are extinct. The current fish fauna is represented by eight orders (Acipenseriformes, Cypriniformes, Cyprinodontiformes, Gobiiformes, Perciformes, Salmoniformes, Siluriformes, and Synbranchiformes) and 18 families (Acipenseridae, Acheilognathidae, Channidae, Cobitidae, Cottidae, Cyprinidae, Esocidae, Gobiidae, Gobionidae, Leuciscidae, Nemacheilidae, Odontobutidae, Percidae, Poeciliidae, Salmonidae, Siluridae, Sisoridae, and Xenocyprididae), of which the largest number of species belong to Leuciscidae (14 species), Nemacheilidae (13 species), and Cyprinidae (9 species). Significant changes in the fish fauna occurred in the 20th century due to anthropogenic factors and climate change. Water diversion for irrigation and construction of canals and reservoirs led to changes in the hydrological regime, which, combined with illegal fishing and overharvesting dramatically affected the abundance of at least eight species and led to the extinction of two, Acipenser nudiventris and Pseudoscaphirhynchus fedtschenkoi. Massive introductions of commercial fish species in the 20th century led to an increase in the alien fauna to 16 species.