Six loci of nucleolar organizer region (NOR) were detected in genomic in situ hybridization (GISH) of cotton (Gossypium). NOR was the characteristic of 45S rDNA but could be generated by genomic DNA (gDNA) extracted from Gossypium species as probe. With twice FISH to the same mitotic cell of G. herbaceum or G. hirsutum, number, position and size for NORs generated from 45S rDNA and gDNA were identified largely similar or even the same. The NORs with gDNA as probe were therefore permanently defined as GISH-NORs. GISH-NORs from G. hirsutum and G. raimondii mitotic images were all terminal types. Four and two GISH-NORs from G. herbaceum (var. africanum) were terminal and centromere types, respectively. Six GISH-NORs in G. hirsutum were chromosome mapped with two in A- and four in D-subgenomes. There were also GISH-NORs in mitotic image of G. raimondii with its own gDNA as probe. From mitotic image of G. herbaceum with its own gDNA as probe, GISH-NOR could not be observed but non-whole- recovery of hybridized signals was distinguished. These non-whole-recovery of hybridized signals were detected on long arm terminals of most chromosomes and especially existed in nearly half long arm of a pair of chromosomes in G. herbaceum gDNA probed itself GISH image, which may be possibly induced by low copy genes within the regions rather than inter-subgenomic segment translocations. GISH-NORs in G. hirsutum mitotic images were dominantly observed when gDNAs from D and A genome species were used as probes and block, respectively, but not when the reverse probe and block gDNA from the two diploid progenitor genomes were designed. There may be two speculations to this special phenomenon: rDNA concerted evolution; content of rDNA in genome D more than genome A.