Background: Cotton (Gossypium spp.), as the world's most utilized textile fibre source, is an important, economically valuable crop worldwide. Understanding the genomic variation of tetraploid cotton species is important for exploitation of the excellent characteristics of wild cotton and for improving the diversity of cotton in breeding. However, the discovery of DNA polymorphisms in tetraploid cotton genomes has lagged behind other important crops. Results: A total of 111,795,823 reads, 467,735 specific length amplified fragment (SLAF) tags and 139,176 highquality DNA polymorphisms were identified using specific length amplified fragment sequencing (SLAF-seq), including 132,880 SNPs and 6,296 InDels between the reference genome (TM-1) and the five tetraploid cotton species. Intriguingly, gene ontology (GO) enrichment analysis revealed that a number of significant terms were related to reproduction in G. barbadense acc. 3-79. Based on the new data sets, we reconstructed phylogenetic trees that showed a high concordance to the phylogeny of diploid and polyploid cottons. A large amount of interspecific genetic variations were identified, and some of them were validated by the single-strand conformation polymorphism (SSCP) method, which will be applied in introgression genetics and breeding with G. hirsutum cv. Emian22 as the receptor and the other species as donors. Conclusions: Using SLAF-seq, a large number of DNA polymorphisms were identified. The comprehensive analysis of DNA polymorphisms provided invaluable insights into the different tetraploid cotton species. More importantly, the identification of numerous interspecific genetic variations provides the basis and is very practical for future introgression breeding. The results presented herein provide a valuable genomic resource for new insights into the genetics and breeding of cotton.