Nucleotide variation in the ACL5 gene region, which encodes spermine synthase, was analyzed for 21 Arabidopsis thaliana ecotypes and one accession of Arabis gemmifera. In A. thaliana, dimorphism was also detected in the ACL5 region, as in other nuclear genes of this plant. The nucleotide diversity (pi) of the entire region, exon and intron was 0.0163, 0.0042 and 0.0293, respectively. The level of nucleotide variation in this region was among the highest of those reported for genes in this plant species. The neutrality tests of Tajima, and Fu and Li did not detect significant deviation from test assumptions for the polymorphism data. However, the HKA test indicated that the level of polymorphism in the intron was significantly high, compared with A. gemmifera. The high nucleotide variation in the intron is responsible for the high level of nucleotide variation in the entire region. These results can be explained by elevated mutation rate in the ACL5 region in the A. thaliana lineage after the two species were split.