Studies on Chenopodium chromosomes are scarce and restricted mainly to chromosome number estimation. To extend our knowledge on karyotype structure of the genus, the organization of 5S and 35S rRNA genes in Chenopodium chromosomes was studied. The rDNA sites were predominantly located at chromosomal termini, except in a few species where 5S rDNA sites were interstitial. The majority of the diploid species possessed one pair each of 35S and 5S rDNA sites located on separate chromosomes. Slightly higher diversity in rDNA site number was observed in polyploid accessions. One or two pairs of 35S rDNA sites were observed in tetraploids and hexaploids. Tetraploid species had two, four or six sites and hexaploid species had six or eight sites of 5S rDNA, respectively. These data indicate that, in the evolution of some polyploid species, there has been a tendency to reduce the number of rDNA sites. Additionally, polymorphism in rDNA site number was observed. Possible mechanisms of rDNA locus evolution are discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ••, ••–••.