a-Expansins are cell wall proteins that facilitate cell wall extension by disrupting noncovalent bonds between wall components. Some of the expansin genes may play a very important role in cell wall extension during fiber development (Harmer et al. 2002; Arpat et al., 2004). The objectives of this study were, (1) to discern the sequence variation of the expansin genes in diploid and tetraploid cotton species; and (2) to find the chromosomal location of these genes in tetraploid cotton. Thirteen pairs of PCR primers were designed to amplify these genes using sequence information of the 6 member of the a-expansin gene family. A total of 104 (13 pairs primer x 8 lines) fragments were successfully amplified from the eight lines, including two diploid species (Gossypium arboreum, G. raimondii), and four tetraploid species, TM-1, RIL-1 and RIL-2 (G. hirsutum), 3-79 (G. barbadense), G. tomentosum, and G. mustelinum. The PCR amplified fragments were purified from agarose gels and cloned into pCR4-TOPO vector by the TA cloning method. Twelve colonies were selected from each of the 104 fragment for forward and reverse sequencing. The alignment of the 96 (12 colonies x 8 lines) amplified fragment, for each pair of PCR primer, was performed with the SeqMan program of DNASTAR software (DNASTAR, Inc., Madison, Wisconsin, USA). Based on the alignment results, each sequence were initial assigned to At or Dt genome by visually comparing the sequence similarity with the sequences in the two diploid species. A phylogenetic tree analysis based on the sequence similarity was conducted by the UPGMA method of PAUP software (Swofford et al., 1993). This phylogenetic tree groupings allowed more precise assignment of each sequence to the At or Dt genome. The sequences of these genes showed limited sequence divergence among the two diploid and 6 tetraploid lines. Comparative analysis of sequence similarity showed differences within and between genomes in the tetraploid cotton species. We are in the process of identifying chromosomal location of these genes with SNP markers using deletion analysis methods with the aneuploid interspecific chromosome substitution stocks. The chromosomal location may facilitate research on candidate gene mapping and possible association of these genes with important fiber development QTLs.