High levels of inter-specific diversity are expected due to genetic isolation, the reproductive or geographical barriers, which lead to the accumulation of nucleotide variation. However, high levels of genetic variation are repeatedly observed even within species, notably at loci of the human major histocompatability complex and of plant resistance genes. Are molecular isolations responsible for the high intra-specific variation? To address this issue, we performed a genome-wide survey of the relationship between the possible factors that could cause genetic isolation, and the level of polymorphism, based on two rice genome comparisons. Here, we show that the levels of polymorphism in rice genes are positively correlated with the proportions of non-alignable flanking sequences, and that the correlation is observed even in single-copy genes. The physical locations of the genes were also investigated, and a strong association between the asymmetric architecture of genomes and the levels of polymorphism was revealed. These results suggest that the flank heterogeneity and the asymmetric architecture between genomes serve as isolation mechanisms at the molecular level that result in accumulation of higher genetic variation. This mechanism is of fundamental importance to understand natural genetic variation within species.