Astyanax bifasciatus Garavelo & Sampaio, 2010 was originally described as endemic to the Iguagu river basin. Between March 2017 and January 2018, specimens of A. bifasciatus were sampled during expeditions to headwater streams of the Piquiri river basin, upper Parana river basin. The identification was confirmed both by morphological and molecular analyses, representing, therefore, the first record of the species outside of the basin of the Iguagu River. In addition, the lack of structuring in the haplotype network confirms that the representatives of A. bifasciatus from both basins appear to comprise a single population. Keywords Distribution range, DNA barcode, endemicity, headwater streams, species identification.