Cotton is a model system for studying polyploidization, genomic organization, and genome-size variation because the allotetraploid was formed 1-2 million years ago, which is old enough for sequence divergence but relatively recent to maintain genome stability. In spite of characterizing random genomic sequences in many polyploidy plants, the cytogenetic and sequence data that decipher homoeologous chromosomes are very limited in allopolyploid species. Here, we reported comprehensive analyses of integrated cytogenetic and linkage maps of homoeologous chromosomes 12A and 12D in allotetraploid cotton using fluorescence in situ hybridization and a large number of bacterial artificial chromosomes that were anchored by simple sequence repeat markers in the corresponding linkage maps. Integration of genetic loci into physical localizations showed considerable variation of genome organization, structure, and size between 12A and 12D homoeologous chromosomes. The distal regions of the chromosomes displayed relatively lower levels of structural and size variation than other regions of the chromosomes. The highest level of variation was found in the pericentric regions in the long arms of the two homoeologous chromosomes. The genome-size difference between A and D sub-genomes in allotetraploid cotton was mainly associated with uneven expansion or contraction between different regions of homoeologous chromosomes. As an attempt for studying on the polyploidy homoeologous chromosomes, these results are of general interest to the understanding and future sequencing of complex genomes in plant species.