Technical Abstract: Upland cotton (G. hirsutum L.) is the leading fiber crop worldwide. Genetic improvement of fiber quality and yield is facilitated by a variety of genomics tools. An integrated genetic and physical map is needed to better characterize quantitative trait loci and to allow for the positional cloning of valuable genes. However, developing integrated genomic tools for complex allotetraploid genomes, like that of cotton, is highly experimental. In this report, we describe an effective approach for developing an integrated physical framework that allows for the distinguishing between subgenomes in cotton. A physical map has been developed with 220 and 115 BAC contigs for homoeologous chromosomes 12 and 26, respectively, covering 73.49 Mb and 34.23 Mb in physical length. Approximately one half of the 220 contigs were anchored to the At subgenome only, while 48 of the 115 contigs were allocated to the Dt subgenome only. Between the two chromosomes, 67 contigs were shared with an estimated overall physical similarity between the two chromosomal homologs at 40%. A total of 401 fiber unigenes plus 214 non-fiber unigenes were located to chromosome 12 while 207 fiber unigenes plus 183 non-fiber unigenes were allocated to chromosome 26. Anchoring was done through an overgo hybridization approach and all anchored ESTs were functionally annotated via blast analysis. This integrated genomic map describes the first pair of homoeologous chromosomes of an allotetraploid genome in which BAC contigs were identified and separated through the use of chromosome-specific probes and locus-specific genetic markers. The approach used in this study should prove useful in the construction of genome-wide physical maps for polyploid plant genomes including Upland cotton. The identification of gene-rich islands in the integrated map provides a platform for positional cloning of important genes and the targeted sequencing of specific genomic regions.