DNA variation in a 4.7-kb region of the cytosolic phosphoglucose isomerase (PgiC) locus was investigated for 21 ecotypes of Arabidopsis thaliana. The estimated nucleotide diversity was 0.0038, which was one-third of those in previously investigated loci. Since most of the nucleotide variations (93%) were singleton and doubleton, Tajima's test statistic was significantly negative. About 50% of nucleotide polymorphisms in exons were replacement, which caused significance in McDonald and Kreitman's test when compared with Arabis gemmifera and Cardaminopsis petraea. These results indicated that DNA polymorphism at the PgiC locus was not under neutrality. There were two divergent sequence types in the PgiC region, which were associated with allozyme variation. The Fast allozyme was shown to have originated from the Slow allozyme, since two outgroup species had the Slow form. A phylogenetic tree of ecotypes with the Fast allozyme had the shape of a star phylogeny. Mismatch distribution of the Fast allozyme ecotypes resembled that expected under an expanding population model. These results suggest positive selection for the Fast allozyme of the PGIC in A. thaliana.