The European bitterlings (Rhodeus amarus) from the Eastern locations were cytogenetically examined by conventional and molecular techniques. All analyzed individuals presented invariably the same chromosomal constitution of 2n = 48, with 8 metacentrics + 20 submetacentrics + 20 subtelo-acrocentrics and C-banding positive heterochromatin at the pericentromeric regions in most of the chromosomes. Moreover, some of the chromosomes had short arms entirely built with heterochromatin. GC-rich Ag-NORs (nucleolus organizer regions) were located at the short arms of two submetacentric chromosomes, and the length polymorphism of these regions was found. Multiple location of 28S rDNA sequences with fluorescence in situ hybridization signals was observed on the long and/or short arms of three submetacentric chromosomes including NOR regions and short arms of three to five acrocentric chromosomes in the studied fish. 5S rDNA sites were found on the short arms of two subtelocentric chromosomes, and telomeric repeats were localized at the ends of all chromosomes. Provided results have expanded our knowledge concerning genetic characteristics of the European bitterlings that may be profitable in the conservation programs of this endangered species.