A PCR-based strategy was employed to identify myb-related genes potentially involved in the differentiation and development of cotton seed trichomes. cDNA clones representing six newly identified cotton myb-domain genes (GhMYB) of the R2R3-MYB family were characterized in the allotetraploid species Gossypium hirsutum L. (2n = 4x = 52; AADD). Several interesting motifs and domains in the transregulatory region (TRR) were identified as potential candidates for modulating GhMYB activity. One such structural feature is a basic 40-amino acid stretch (TRR1) located immediately downstream of the DNA-binding domain (DBD) in five of the GhMYBs. Furthermore, the conserved motif GIDxxH identified in a subset of plant MYBs is also present in the same position in the TRR1 domains of GhMYB1 and GhMYB6, exactly 12 amino acid residues downstream of the last tryptophan in the R3 repeat of the DBD. At least two of the GhMYBs (GhMYB4 and GhMYB5) contain unidentified ORFS in the 5' leader sequence (5'-uORFs) that may serve to regulate the synthesis of these particular GhMYB proteins at the translational level. Multiple alignment of DBD sequences indicated that GhMYBs show structural similarity to plant R2R3-MYB factors implicated in phenylpropanoid biosynthesis. GhMYB5 is the most distantly related cotton R2R3-MYB and is found in an isolated cluster that includes the drought-inducible AtMYB2. Sequence comparisons of DBD and TRR domains from GhMYBs, MIXTA (AmMYBMx) and G11 (AtMYBG11) did not reveal any striking similarity beyond conserved motifs. However, based on earlier phylogenetic analysis, GhMYB2, GhMYB3, and GHMYB4 are members of a cluster that contains GLABROUS1, while GhMYB1 and GhMYB6 belong to a closely related cluster. Semi-quantitative RT-PCR analysis revealed two discrete patterns of GhMYB gene expression. Type I cotton MYB (GhMYB-1, -2, and -3) transcripts were found in all tissue-types examined and were relatively more abundant than those derived from type II GhMYB genes (GhMYB-4, -5, and -6), which showed distinct, tissue-specific expression patterns. The developmental regulation of GhMYBs is consistent with a role for these DNA-binding factors in the differentiation and expansion of cotton seed trichomes.