The subfamily Stethaprioninae, a monophyletic group among characids, is almost unknown from the cytogenetic viewpoint. This paper shows the first chromosome characterization of Poptella paraguayensis, an endemic species of the Paraguay river basin. Analysis in Giemsa-stained preparations showed 2n=50: 10M + 26SM + 8ST + 6A. The nucleolar organizing regions (NORs) detected by silver nitrate and GC-specific fluorochrome mithramycin (MM) staining showed two NOR-bearing chromosomes in the diploid complement. DA/DAPI staining showed no differential fluorescence along the chromosomes. C-banding evidenced small blocks of heterochromatin in the centromeric and telomeric regions. Phylogenetic relationships between the Stethaprioninae and other characids are discussed on the base of chromosome data.