The genus Notionotus Spangler, 1972 has a wide distribution in the Neotropical and Oriental region. Species of this genus are characterized by their small size and occurrence in streams and seepages. The last revision of the genus for the Neotropical region supports 18 species, of which 12 are new species. In this study mitochondrial and nuclear genes COI, H3, CAD, 18S and 28S are used to reconstruct the phylogeny of Notionotus in the Neotropics. According to the collected data, it is predictable that the genus Notionotus is not monophyletic, however Notionotus in the Neotropics are monophyletic. The phylogeny showed that Neotropical species of Notionotus are divided into two groups liparus and lohezi, and there are morphological characters to diagnose both groups of species. The shape of the mesoventrite and the male genitalia can be used to diferentiate species group. lohezi species groups has a clear distribution in the Guiana Shield while liparus species groups is widely distributed in Central America and the Northern of South America.