Technical Abstract: Informative, abundant, high-throughput markers associated with genes such as SNPs or insertion/deletions (indels) are desirable for both breeding and genetic analyses. Expressed genes are available as templates to study variation. In G. arboreum, 24,597 non-redundant transcripts are available; with 27,355 in G. raimondii; and 63,138 in G. hirsutum. The current report describes a strategy to efficiently identify and characterize SNP markers that are useful to manipulate and transfer novel alleles to breeding germplasm in cotton. Different DNA templates were evaluated for their specificity to amplify single-copy loci, and polymorphism within and among species, with emphasis on cultivated cotton. We demonstrate that single-copy loci can be efficiently amplified in cotton despite redundancy conferred by its allopolyploid origin, and that they can be mapped to specific genomes. In this research, SNP and indel diversity is characterized for 270 single-copy polymorphic loci in cotton. The strategy for SNP discovery is described to pre-screen loci for copy number and polymorphism. Diversity was characterized in a broad set of breeding lines and exotic lines representing a standard germplasm panel indicating that G. barbadense is much more diverse than G. hirsutum. Our data also indicate that the A and D genomes in both diploid and tetraploid cotton remain distinct from each other such that homoeologs can be distinguished.