Gossypium mustelinum((AD)_4) is one of five disomic species in Gossypium.Three 45S ribosomal DNA (rDNA) loci were detected in(AD)_4 with 45S rDNA as probe,and three pairs of brighter signals were detected with genomic DNA(gDNA) of Gossypium D genome species as probes.The size and the location of these brighter signals were the same as those detected with 45S rDNA as probe,and were named GISH-NOR.One of them was super-major,which accounted for the fact that about one-half of its chromosome at metaphase was located at chromosome 3,and other two were minor and located at chromosomes 5 and 9, respectively.All GISH-NORs were located in A sub-genome chromosomes,separate from the other four allopolyploid cotton species.GISH-NOR were detected with D genome species as probe,but not A.The greatly abnormal sizes and sites of(AD)_4 NORs or GISH-NORs indicate a possible mechanism for 45S rDNA diversification following(AD)_4 speciation.Comparisons of GISH intensities and GISH-NOR production with gDNA probes between A and D genomes show that the better relationship of(AD)_4 is with A genome.The shortest two chromosomes of A sub-genome of G.mustelinum were shorter than the longest chromosome of D sub-genome chromosomes.Therefore,the longest 13 chromosomes of tetraploid cotton being classified as A sub-genome,while the shorter 13 chromosomes being classified as D sub-genome in traditional cytogenetic and karyotype analyses may not be entirely correct. Gossypium mustelinum ((AD) _4) is one of five disomic species in Gossypium.Three 45S ribosomal DNA (rDNA) loci were detected in (AD) _4 with 45S rDNA as probe, and three pairs of brighter signals were detected with genomic DNA gDNA) of Gossypium D genome species as probes. size and the location of these brighter signals were the same as those detected with 45S rDNA as probe, and were named GISH-NOR. One of them was super-major, which accounted for the fact that about one-half of its chromosome at metaphase was located at chromosome 3, and the other two were minor and located at chromosomes 5 and 9, respectively. All GISH-NORs were located in A sub-genome chromosomes, separate from the other four allopolyploid cotton species. GISH-NOR were detected with D genome species as probe, but not A. The major abnormal sizes and sites of (AD) _4 NORs or GISH-NORs indicate a possible mechanism for 45S rDNA diversification following (AD) _4 speciation Comparisons of GISH intensities and GISH-NOR production with gDN A probes between A and D genomes show that the better relationship of (AD) _4 is with A genome. The shortest two chromosomes of A sub-genome of G. mustelinum were shorter than the longest chromosome of D sub-genome chromosomes.Therefore, the longest 13 chromosomes of tetraploid cotton being classified as A sub-genome, while the shorter 13 chromosomes being classified as D sub-genome in traditional cytogenetic and karyotype analyzes may not be entirely correct.