Double-color fluorescence in situ hybridization (FISH) followed by DAPI counterstaining allowed the chromosomal assignment of 5S and 18S-25S rRNA genes in the four cultivated Phaseolus Species; P. vulgaris, P. coccineus, P. acutifolius, and P. lunatus (all: 2n = 2x = 22). The rRNA gene loci display variation between species as reflected in differences of signal size and (or) number. From one to three pairs of 5S sites and one to seven pairs of 18S-25S sites were found in the diploid complements of the four taxa studied. Intraspecific variation was studied in P. vulgaris, and it is shown that the number of 18S-25S rDNA sites differs between cultivars. Cytogenetic mapping was complemented by karyotype analyses. Each of the four cultivated Phaseolus species exhibits a characteristic heterochromatin endowment, with P. acutifolius var. latifolius having the highest amount of C-band material. Quantitative karyotyping in combination with cytogenetic mapping allowed the identification of homeologous chromosomes in the different species.