DNA variation was studied in a 2.2 kb region of the regulatory gene Atmyb2 using 20 ecotypes of Arabidopsis thaliana and one accession each of Arabis gemmifera and Arabidopsis himalaica. Nucleotide diversity (π) in the region was 0.0027, which was lower than for other loci in A. thaliana. The MYB domain of the Atmyb2 gene (π = 0.0036) had a larger variation than the non-MYB region (π = 0.0013). Tajima's test and Fu and Li's test did not give a significant result. In contrast to the low level of polymorphism, the degree of divergence of the Atmyb2 region was higher between A. thaliana and A. gemmifera (K = 0.0730) than for other loci. The MYB domain (K = 0.0436) had smaller divergence than the non-MYB region (K = 0.0939). The HKA test detected significant discordance in the ratio of polymorphism to divergence in some comparisons. The pattern of low polymorphism and high divergence, which is mainly observed in the non-MYB region of the gene, is inconsistent with the neutral mutation theory. Strong purifying selection after establishment of A. thaliana and a species-specific adaptive process could be invoked to account for this pattern of polymorphism and divergence of Atmyb2.