The evolution of genome size and ribosomal DNA (rDNA) locus organization was analysed in 23 diploid species of Chenopodium s.l., all of which share the same base chromosome number of x = 9. Phylogenetic relationships among these species were inferred from plastid and nuclear ribosomal internal transcribed spacer (nrITS) DNA sequences. The molecular phylogenetic analyses assigned all analysed species of Chenopodium s.l. to six evolutionary lineages, corresponding to the recent new generic taxonomic treatment of Chenopodium s.l. The distribution of rDNA loci for four species is presented here for the first time using fluorescence in situ hybridization (FISH) with 5S and 35S rDNA probes. Most of the 23 analysed diploid Chenopodium spp. possessed a single subterminally located 35S rDNA locus, except for three species which possessed two 35S rDNA loci. One or two 5S rDNA loci were typically localized subterminally on chromosomes, rarely interstitially. Analyses of rDNA locus numbers in a phylogenetic context resulted in the reconstruction of one locus each of 35S rDNA and 5S rDNA, both in subterminal positions, as the ancestral state. Genome sizes determined using flow cytometry were relatively small (2C value < 2.8 pg), ranging from 0.734 pg in C. schraderianum to 2.721 pg in C. californicum (nearly four-fold difference), and were often conserved within major phylogenetic lineages, suggesting an adaptive value. The reconstructed ancestral genome size was small for all evolutionary lineages, and changes have probably coincided with the divergence of major lineages.