This study examines the relationship between DNA sequence variation and level of gene expression in four metallothionein genes from wild rice Oryza rufipogon. The nucleotide diversity was 0.0028 to 0.0117 over the entire coding and non-coding region, and it was negatively correlated with gene expression for three type 2 metallothionein genes. In contrast, codon bias and percent of preferred codons correlated positively with gene expression. These results indicate that the intensity of natural selection depends on the level of gene expression, which in turn shapes the level of nucleotide polymorphism. In addition, significant linkage disequilibria were frequent between the metallothionein genes, although significance was not confirmed after multiple test correction. This result suggests that metallothionein genes expressed at different levels are epistatic with respect to fitness, and that gene expression is an important factor determining level of DNA polymorphism.