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We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM (∼600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM (∼500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6–11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the “portability” of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.