The most widely cultivated species of cotton,Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome speciesG. herbaceum andG. arboreum, the D-genome speciesG. raimondii andG. thurberi, and the AD tetraploidG. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S–28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six newG. hirsutum 18S–28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybrizization signal at these loci allowed us to designate them as major, intermediate, or minor 18–28S loci. Using genomic painting with labeled A-genome DNA, five 18S–28S loci were localized to theG. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S–28S rDNA loci inG. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species has three loci and both D-genome species had four.G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S–28S rDNA loci. All four of the diploid genomes wer examined contained a single 5S locus. InG. herbaceum (A1) andG. thurberi (D1), the 5S locus is syntenic to a major 18S–28S locus, but inG. arboreum (A2) andG. raimondii (D5), the proposed D-genome progenitor ofG. hirsutum, the 5S loci are syntenic tominor and intermediate 18S–28S loci, respecitively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S–28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repearts can differ widely.