Scinax alter, a taxon belonging to the S. ruber clade, has been previously suggested to represent a species complex. We analyzed variation among populations of Scinax alter using advertisement calls, dorsal color pattern, and external morphology. We identified three diagnosable groups distributed throughout the Atlantic Forest of eastern Brazil, which differ mainly in the advertisement call, dorsal drawing pattern, snout–vent length, and presence of tubercles on tarsus. Scinax alter was restricted to populations from south of Bahia State to Rio de Janeiro State, and two new species were related to the southern populations: Scinax imbegue, from Parque das Nascentes, Municipality of Blumenau (27°03′S, 49°05′W, 412 m a.s.l.), Santa Catarina State, Brazil, and Scinax tymbamirim, from Córrego Grande (27°35′S, 48°31′W, at sea level), Municipality of Florianópolis, Santa Catarina State, Brazil.