ABSTRACT The genus Luteostriata Carbayo, 2010, currently comprises 10 species distributed in southeastern and southern Brazil. Species of this genus represent a taxonomic challenge because many have very similar external and internal features. Some species were described based on specimens from many localities using anatomical and histological features without phylogenetical analyses, such as Luteostriata ernesti, known from various localities in southeastern and southern Brazil. We conducted an integrative taxonomic approach of southeastern and southern specimens of L. ernesti, performing morphological analyses and using two molecular markers. Our results reveal that there are at least two clades (southeastern and southern) under the designation L. ernesti, and phylogenetic analyses show that they are not sister groups. Thus, we took the taxonomic decision to describe a new species, Luteostriata dissimilis Iturralde & Leal-Zanchet, sp. nov., which is split off from L. ernesti based on specimens of the southern clade. The phylogenetic analyses suggest a close relationship between Luteostriata dissimilis and Luteostriata muelleri and indicate that Luteostriata ernesti is more closely related to Luteostriata caissara. Our results also indicate that species delineation may be the first approach to understanding the diversity and distribution of species in this genus and that a complete taxonomic revision of Luteostriata species is necessary. http://www.zoobank.org/urn:lsid:zoobank.org:act:F83EE656-A491-493D-9F25-97413097D007