The taxonomic status of gudgeons (Gobionidae) and the genetic relationships among them are still unclear. The current knowledge on the molecular cytogenetics of gudgeons is quite limited and concerns the basic parameters of their karyotype. Therefore, the main aim of this study was to obtain new data on the chromo- somes of two gudgeons – Gobio gobio and Romanogobio belingi – which would allow for characterising these species in a more precise way. Chromosomes of the examined species were analysed by conventional Giemsa staining and, for the first time, by fluorescence in situ hybridisation (FISH) with 28S rDNA, 5S rDNA and telomeric probes. The diploid chromosome numbers in both species were the same and equalled 2n=50, while the chromosome arm number (NF) was 96 and 98 in G. gobio and R. belingi, respectively. Moreover, small interspecific differences concerning the chromosomal morphology were observed. Major 28S rDNA sites were mapped to the short (p) arms of two submetacentric chromosomes with a similar size in G. gobio, whereas in R. belingi, FISH signals were observed from the short and the long (q) arms of two different-sized submetacentric chromosomes. Minor 5S rDNA sites were found on the p arms of eight submetacentric chromosomes of different sizes in G. gobio and on five submetacentric chromosomes in R. belingi. Unique chromosomal co-locations of major and minor rDNA sites were observed in the karyotypes of both species. In the chromosomes of both species, telomeric DNA sequences were typically located at the ends of all chromosomes. The number of chromosome arms, as well as the number and lo- cation of 45S and 5S rDNAs, and the number of unexpected co-located major and minor ribosomal genes may be useful cytotaxonomic species-specific markers.