Triploid viviparous onions [Allium x cornutum Clementi ex Visiani 1842, syn Allium cepa L. var. viviparum Metzg. (Alef.), auct.] (2n = 3x = 24), are known in some countries only as rare relict crops. In other parts of the world they are still traditionally or even commercially cultivated. In previous cytogenetic studies of the Croatian triploid viviparous onion Ljutika, Giemsa C-banding, chromosome pairing analysis during meiosis, and genomic hybridization in situ indicated a complex hybrid with highly heterozygous karyotype structure, with possible triparental genome organization. This study continues an analysis of the karyotype structure of Ljutika. Staining with fluorochromes CMA3 (Chromomycin A3) and Dapi (4,6-diamidino-2-phenylindole) confirmed previous results from Giemsa C-banding and revealed GC-rich heterochromatic regions associated mainly with chromosome ends and nucleolus organizing regions (NORs), and only a few interstitial bands. Fish mapping of the ribosomal 18S-5.8S-26S genes revealed two major rDNA signals on the short arms of two subtelocentric satellite chromosomes in almost all metaphase plates of Ljutika. The largest subtelocentric chromosome lacked rDNA signals. A significantly smaller rDNA signal was occasionally located on one small submetacentric chromosome. These results are in agreement with previously published results from identification of NORs by silver-staining technique, which confirmed a maximum three nucleoli in interphase nuclei. We discuss the molecular mechanisms underlying rearrangements and activity of ribosomal genes in the triploid karyotype.