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The fish fauna from the Lower Iguaçu River and tributaries upstream of the Iguaçu Falls, the last free-flowing river stretch, were investigated. Twenty five sites in tributaries and the main channel were sampled between 2010 and 2016 using several kinds of fishing gear. The species were categorized according to their size, origin, and conservation status. Species richness and abundance in the main channel and tributaries were compared. In total, 87,702 specimens were recorded, comprising 76 species, 25 families, 53 genera, and eight orders. Characiformes and Siluriformes were the richest orders, representing 92% of the total specimens; Characidae, Cichlidae, Pimelodidae, and Loricariidae were the richest families. The fish fauna was composed of small and medium-sized species and included endemic (42%), autochthonous (24%), allochthonous (21%), and exotic (9%) species, as well as hybrids (4%). Significant differences in the relative numerical abundance of species were found among sites. Ancistrus mullerae and Rhamdia branneri (endemic) were indicator species for tributaries inside of Iguaçu National Park (INP), while Phalloceros harpagos (autochthonous) and Ictalurus punctatus (exotic) for tributaries outside of INP and Odontesthes bonariensis (allochthonous) for the main channel. The last dam-free stretch of the Lower Iguaçu River and tributaries upstream the Iguaçu Falls exhibits a rich endemic fish fauna, including some rare, endangered species ( Steindachneridion melanodermatum , Gymnogeophagus taroba , and Psalidodon gymnogenys ). These findings are essential to predict and understand the effects caused by the new Baixo Iguaçu Hydroelectric Power Plant and highlight the importance of tributaries and Iguaçu National Park for conservation of endemic species., (Suelen Fernanda Ranucci Pini, Maristela Cavicchioli Makrakis, Mayara Pereira Neves, Sergio Makrakis, Oscar Akio Shibatta, Elaine Antoniassi Luiz Kashiwaqui.)