The karyotypes of Allium , a genus that comprises many crops and ornamental plants, are relatively poorly studied. To extend our knowledge on karyotype structure of the genus, the chromosomal organization of rRNA genes and CMA/DAPI bands was studied. Fluorescence in situ hybridization using 5S and 35S rDNA probes and banding methods (silver staining and CMA 3 /DAPI staining) were used to analyze the karyotypes of eight cultivated Allium L. species. Analyzed Allium taxa revealed three different basic chromosome numbers ( x = 7, 8, 9) and three different ploidy levels (diploid, triploid, and tetraploid). The rDNA sites chromosomal organization is reported the first time for the six species ( A . moly , A . oreophilum , A . karataviense , A . nigrum , A . sphaerocephalon , A . porrum ). The Allium species that were analyzed showed a high level of interspecies polymorphism in the number and localization of the rDNA sites. The fluorescence in situ hybridization patterns of 35S rDNA sites were more polymorphic than those of the 5S rDNA in the diploid species. Several groups of similar chromosomes could be distinguished among the chromosomes that had rDNA sites in the polyploid species. Each of the groups had three chromosomes (triploid A . sphaerocephalon L.) or four chromosomes (tetraploid A . porrum L.) suggesting their autopolyploid origin. In the genomes of four of the analyzed species, only some of the 35S rDNA sites were transcriptionally active. Fluorochrome banding revealed that the CMA 3 + bands were associated with the 35S rDNA sites in all of the species that were analyzed, except A . fistulosum L. in which positive CMA 3 + bands were detected in the terminal position of all of the chromosome arms. The rDNA sequences, nucleolar organizer regions (NORs), and CMA/DAPI bands are very good chromosome markers that allowed to distinguished from two to five pairs of homologous chromosomes in analyzed Allium species. The karyotypes of the studied species could be clearly distinguished by the number and position of the rDNA sites, NORs, and CMA/DAPI bands, which revealed high interspecific differentiation among the taxa. Electronic supplementary material The online version of this article (10.1007/s13353-018-0474-1) contains supplementary material, which is available to authorized users.