MYB transcription factors represent a family of proteins which include a conserved domain, the MYB DNA-binding domain. The MYB proteins bind to DNA and regulate gene expression. In plants, MYB protein subfamilies are involved in many physiological and biochemical processes including regulation of trichome length and density. It has been reported that the developmental regulations of some MYB transcription factors are involved in cotton fiber differentiation and expansion. PCR primer pairs specific to the six MYB genes (MYB1, MYB2, MYB3, MYB4, MYB5 and MYB6) had been used for amplification of genomic DNAs from five different tetraploid cotton lines, HS46 and MARCABUCAG8US-1-88 (Gossypium hirsutum), 3-79 (G. barbadense), G. tomentosum, and G. mustelinum. Each amplicon was cloned and sequenced by following the standard protocol and the sequences were analyzed with DnaSP 4.0 software (Rozas et al., 2003). The duplicated homologue sequences of the tetraploid lines for each MYB gene were identified by aligning with the according sequences of diploid A and D genome ancestral species (G. herbaceum and G. raimondii) and the sequences of TM-1 (G. hirsutum) homologue sequences from GeneBank database. The phylogenetic trees were constructed based on the rooted parsimony algorithm of the software package MEGA 3.1 (Kumar et al., 2004). The putative designation of any tetraploid sequence to a locus or genome was based on the results of phylogenetic tree and its relationship with the homologue sequence of the two diploid ancestral species. SNPs were discovered based on the comparative analysis of the aligned sequences at a putative locus. Phylogram results showed that the sequences of tetraploid species for each of the MYB genes fall into two broad groups, each containing one of the two homeologous sequences from the tetraploid lines and the corresponding copy from the two ancestral diploid species. Results suggested that the duplicated MYB genes have evolved independently of one another following polyploidy formation, which confirmed the results of earlier studies. The strategy of identifying chromosomal locations of SNP markers was followed the overall method of deletion analysis using both aneuploid and euploid substitution lines. The SNaPshot MultiPlex Kit (Applied Biosystems, Foster City, CA) was used to detect SNP markers according to the manufacturer's protocol. Fifteen different interspecific (TM-1 and 3-79 or TM-1 and G. tomentosum) SNP primers specific to different MYB genes were used for chromosomal assignment of these markers. Results showed that the chromosomal locations of MYB4 were on the long arm of chromosomes 7 and 16 confirming their homeologous relationship. Gene MYB1, MYB2 and MYB5 were assigned to 18Lo, 8sh and 11sh, respectively. The detail results of SNPs characterization, nucleotide diversity and haplotype analysis of the six MYB genes will be presented in the conference.