This chapter provides information on ontogenetic patterns of neotropical fish species distribution in tributaries (Verde, Pardo, Anhanduí, and Aguapeí rivers) of the Porto Primavera Reservoir, in the heavily dammed Paraná River, Brazil, identifying key spawning and nursery habitats. Samplings were conducted monthly in the main channel of rivers and in marginal lagoons from October through March during three consecutive spawning seasons in 2007-2010. Most species spawn in December especially in Verde River. Main river channels are spawning habitats and marginal lagoons are nursery areas for most fish, mainly for migratory species. The tributaries have high diversity of larvae species: a total of 56 taxa representing 21 families, dominated by Characidae. Sedentary species without parental care are more abundant (45.7%), and many long-distance migratory fish species are present (17.4%). Migrators included Prochilodus lineatus, Rhaphiodon vulpinus, Hemisorubim platyrhynchos, Pimelodus maculatus, Pseudoplatystoma corruscans, Sorubim lima, two threatened migratory species: Salminus brasiliensis and Zungaro jahu, and one endangered migratory species: Brycon orbignyanus. Most of these migratory species are vital to commercial and recreational fishing, and their stocks have decreased drastically in the last decades, attributed to habitat alteration, especially impoundments. The fish ladder at Porto Primavera Dam appears to be playing an important role in re-establishing longitudinal connectivity among critical habitats, allowing ascent to migratory fish species, and thus access to upstream reaches and tributaries. Establishment of Permanent Conservation Units in tributaries can help preserve habitats identified as essential spawning and nursery areas, and can be key to the maintenance and conservation of the fish species in the Paraná River basin.