R2R3-MYB transcription factors have been implicated in a diversity of plant-specific processes. Among the functions attributed to myb factors is the determination of cell shape, including regulation of trichome length and density. Because myb transcription factors are likely to play a role in cotton fiber development, the molecular evolutionary properties of six MYB genes previously shown to be expressed in cotton fiber initiation were examined. In accordance with their presumed central role, each of the genes display conservative substitution patterns and limited sequence divergence in diploid members of the genus Gossypium, and this pattern is conserved in allotetraploid cottons. In contrast to highly reiterated rDNA repeats, GhMYB homologues (duplicated gene pairs) exhibit no evidence of concerted evolution, but instead appear to evolve independently in the allopolyploid nucleus. Expression patterns for the MYB genes were examined in several organs to determine if there have been changes in expression patterns between the diploids (G. raimondii and G. arboreum) and the tetraploid (G. hirsutum) or between the duplicated copies in the tetraploid. Spatial and temporal expression patterns appear to have been evolutionarily conserved, both during divergence of the diploid parents of allopolyploid cotton and following polyploid formation. However, the duplicated copies of MYB1 in the tetraploid are not expressed at equal levels or equivalently in all organs, suggesting possible functional differentiation.