The chromosomes of the two species of needlefish,Potamorraphiscf.eigenmanniandStrongylura microps, from fresh waters of Argentina were analyzed using C‐banding and silver staining and their karyotypes were described for the first time. The former species possessed 2n‐54 and karyotype composed of three pairs metacentric to submetacentric chromosomes and 24 pairs of subtelo to telocentric chromosomes. The NORs were situated on the end of the shorter arms of one middle‐sized bi‐armed pair of chromosomes. The later species possessed 2n=50 and karyotype composed exclusively of subtelo to telocentric chromosomes gradually decreasing in size. The NORs were situated inter‐stitially on the third pair of chromosomes. One or two supernumerary chromosomes were observed in one individual of the sample. Present data indicate greater cytotaxonomic diversity among species of needlefish which might be associated with their colonization of fresh waters.