Karyotype and chromosomal characteristics of both minor and major rDNA of Sternopygus macrurus, a weakly electric South American fish, from two populations of the upper Paraná River basin, were investigated using conventional (Giemsa staining, silver staining, C-banding and base-specific fluorochromes) and molecular (fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes) cytogenetic techniques. Diploid chromosome number was invariably 2n = 46 and karyotype composed of 23 pairs of biarmed chromosomes (28m+18sm). The nucleolus organizer regions (NORs) were located in the secondary constriction of the p arm of pair No. 2; this site corresponded with CMA3 positive as well as with 18S rDNA signals, respectively. This 18S rDNA cluster was not syntenic to the 5S rDNA sites located at pairs Nos. 1, 5 and 15. The karyotypes and other chromosomal characteristics of individuals from the two populations in the upper Paraná River basin were identical. The karyotype differences among individuals identified as S. macrurus from Paraná River and the São Francisco and Amazon River basins, respectively, may indicate that these taxa might represent distinct species.