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Ituglanis australis new species, is described from tributaries of the Laguna dos Patos and Río Uruguay, in Brazil and Uruguay. This represents the southernmost record of the genus and the first occurrence of a species of Ituglanis in those systems. It is distinguished from all its congeners, except Ituglanis parahybae and Ituglanis cahyensis, by its body pigmentation with three well-defined dark brown stripes running along each flank. Ituglanis australis differs from I. parahybae and I. cahyensis in the pectoral- and pelvic-fin ray counts, the pattern of the cephalic laterosensory system and the number of dorsal-fin basal radials. The new species, as well as several other examined congeners, has the levator internus IV muscle attached to the dorsal face of the posttemporo-supracleithrum; a condition that corroborates the inclusion of Ituglanis into a large trichomycterine clade that also includes Bullockia, Hatcheria, Scleronema and several species of Trichomycterus. Previous proposals of the affinities within Ituglanis are reviewed and, despite some advances, the phylogenetic relationships among species of the genus remain largely unknown. [ABSTRACT FROM AUTHOR]