Neotropical stream fishes exhibit a complex evolutionary history and encompass both old and recent lineages. Patterns of species diversity of stream fishes are relatively well-studied for Neotropical streams, but not for patterns of clade distribution and historical factors that structure these assemblages, which are the main interests of phylogenetic ecology. Understanding the evolutionary context of communities provides important insights into large-scale mechanisms that structure them. This review aims to: (i) discuss the main concepts of phylogenetic ecology and its application to Neotropical stream fishes; and (ii) highlight the main methods applied in this background. The first section presents the main phylogenetic hypothesis of fishes and discusses how their gaps in Neotropical stream fishes hinder phylogenetic ecology. Afterward, we discuss the main concepts of phylogenetic ecology (phylogenetic signal, community phylogenetic structure, and phylogenetic diversity), as well as gaps and potential applications of these concepts and tools to understand Neotropical stream fish assemblages. The second section introduces the main methods to address the phylogenetic ecology, including a standardized procedure to edit fish phylogenetic trees, comparative methods, and indices and analytical tools to understand community structure and conservation importance. Finally, we discuss the perspectives to the next years to better understand the Neotropical stream fish assemblages in the light of past and current historical processes.