It has been suggested that evolutionary changes in regulatory genes may be the predominant molecular mechanism governing both physiological and morphological evolution. R2R3-AtMYB is one of the largest transcription factor gene families in Arabidopsis. Using inferred ancestral sequences we show that several lineages in the R2R3-AtMYB phylogeny experienced excess non-synonymous nucleotide substitution upon gene duplication, indicating episodes of positive selection driving adaptive shifts early in the evolution of this gene family. A noise reduction technique was then used to determine individual sites in DNA-binding domains (R2 domain and R3 domain) of R2R3-AtMYB protein sequence that were favored by frequent non-synonymous substitutions. The analyses reveal that the first helix (helix1) and the second helix (helix2) in both R2 and R3 domains are characterized by more frequent non-synonymous substitutions, and thus experienced significantly higher positive selection pressure than the third helix (helix3) in both domains. Previous MYB protein structure studies have suggested that helix1 and helix2 in both R2 and R3 domains are involved in the characteristic packing of R2R3-AtMYB DNA-binding domains. This suggests that excess non-synonymous substitutions in these helices could have resulted in MYB recognition of novel gene target sites.