AbstractDeuterodonis a genus of the subfamily Stethaprioninae, a group of Neotropical fish known as tetras.Deuterodon hastatusrepresents a species complex, which is supported by cytogenetic and molecular data. In this study, we show the results of comparative evolutionary analyses of theATP synthase subunit 6gene in fourDeuterodonspecies, in addition to ribosomal markers (18S rDNAand5S rDNA), of a new population of theD. hastatusspecies complex from the Angra dos Reis/RJ region. The study population comprised a new cytotype, which we refer to as cytotype D, inD. hastatus, with 2n = 50 = 6M+8SM+8ST+28A. We obtained three different clades ofD. hastatusin our phylogeny, one of them composed only by specimens of cytotype D. By using molecular clock dating, we observed that the radiation ofDeuterodonfrom southeastern Brazil seemed to be associated with neotectonic events that occurred during the Miocene-Pliocene and Pliocene-Pleistocene transitions, marked by the capture of headwater streams and marine transgressions. The results obtained reinforce the idea thatD. hastatusis a species complex, and at least three evolutionary significant units were identified in this group.