Scinax is the most species-rich genus of Neotropical treefrogs, with 129 currently recognized species divided between two major clades, the S. catharinae and S. ruber clades. The S. catharinae clade includes 52 species currently placed in the S. perpusillus and S. catharinae groups, whereas the S. ruber clade is composed of 77 species, 13 of which are included in two species groups: the S. rostratus and S. uruguayus groups, with all 64 remaining species being unassigned to any group. Although some studies have addressed the phylogenetic relationships of the genus, its relationships remain poorly understood. To test the monophyly of the genus, its major clades, and the currently recognized species groups, and the relationships within and among them, we performed a total evidence phylogenetic analysis including sequences of five mitochondrial (portions of cytochrome b and cytochrome oxidase c subunit I, and 12S rRNA, 16S rRNA, NADH dehydrogenase subunit 1, plus three intervening tRNAs) and six nuclear genes (portions of chemokine receptor type 4, proopiomelanocortin, seven in absentia homolog 1, recombination activating gene 1, rhodopsin exon 1, and tyrosinase), and 159 phenotypic characters. The dataset included 121 of the 129 known species of Scinax and many unnamed species. Most species are represented by multiple specimens, including topotypic material for approximately 52% of the species. As a result of this analysis, we partition Scinax into three genera. We restrict Scinax to most species of the former S. ruber clade and divide its species among 13 species groups: the S. auratus, S. boesemani, S. cruentomma, S. danae, S. elaeochroa, S. eurydice, S. funereus, S. fuscomarginatus, S. fuscovarius, S. granulatus, S. nasicus, S. rostratus, and S. squalirostris groups; only one species (S. pachycrus) remains unassigned to any group. To eliminate the paraphyly of Scinax, we redefine Julianus—originally erected for the S. uruguayus group—to include the former S. camposseabrai. We recognize Ololygon for the species of the former S. catharinae clade and divide its species among seven species groups: the O. agilis, O. argyreornata, O. belloni, O. cardosoi, O. catharinae, O. feioi, and O. perpusilla groups. All species groups of the three recognized genera of Scinaxini are discussed, diagnosed, characterized—in many cases using phenotypic synapomorphies—and taxonomic comments are provided for many species. Our study further revealed 57 candidate species, an increase of 44.2% of the recognized species in the tribe, highlighting how far we are from fully understanding the diversity of this clade of Neotropical treefrogs.