Lycopersicon peruuianum shows a gametophytic self-incompatibility (GSI). GSI is controlled by a single locus (S locus) with multiple alleles. S ribonucleases encoded in S alleles cosegregate with their phenotypes of GSI in genetic cross. To understand the genetic role of S allele in L, peruuianum, two large genomic fragments isolated previously were analyzed with total genomic DNAs from several tomato lines generated by cross-pollination. Southern blot analysis with the S allele fragments as probes revealed that the flanking region of S allele contained the highly homologous regions. It is speculated that they may play an important role to prevent genetic cross by self-pollination.