Cotton (Gossypium hirsutum L.) fibers are derived from ovule epidermis, which are developmentally similar to Arabidopsis trichome where several MYB transcription factors have been shown to control their formation. However, little is known about the molecular control of cotton fiber initiation. Here we isolated 55 cotton MYB domain-containing sequences expressed in ovules during fiber initiation. Among them, GhMYB109, encoding a R2R3 MYB transcription factor of 234 amino acids, was found to be structurally related to AtMYBGL1 and AtWER controlling the trichome initiation in Arabidopsis thaliana. Southern blot hybridization revealed that GhMYB109 is present as a unique-copy gene in cotton genome. RNA expression analysis showed that it is specifically expressed in cotton fiber initial cells as well as elongating fibers. These results suggested that GhMYB109 likely plays a direct role in the initiation and elongation of cotton fiber cells.