The species of the family Prochilodontidae have a stable karyotype, made of 54 biarmed chromosomes, and one pair bearing the nucleolus organizer regions (NORs). Despite its considerable economic importance in fluvial basins, no cytogenetic data are available for the Argentinian populations of the species Prochilodus lineatus (Valenciennes, 1847). Forty five P. lineatus specimens from the middle Paraná River (Santa Fe City, Argentina) were analyzed cytogenetically. Chromosome preparations were obtained by kidney cell suspension analyzed directly or after short-term culture, and processed for Giemsa staining, C-and NOR-banding. Fluorescence In Situ hybridization with a 28S rDNA probe was also performed. All specimens exhibited the expected familiar karyotypic features, with 2n = 54 and fundamental number of chromosomes (FN) = 108, and C-banding pattern. Silver staining and FISH detected the NORs on a single chromosome pair. In spite of this invariable condition, positional differences on the NORs-bearing chromosomes were observed. Seventy percent of the specimens showed interstitial NORs, while the remaining 30% presented telomeric positive bands.