Amylose content, an important determinant of rice starch quality, is primarily controlled by the Waxy (wx) gene encoding Granule-Bound Starch Synthase (GBSS). Oryza rufipogon, known as the ancestor of Asian cultivated rice (O. sativa L.), is the most important wild relative for rice improvement. To obtain basic information on the diversity of wx microsatellite alleles and on the relationship between wx microsatellite alleles and amylose classes, 212 strains of wild rice were collected and analyzed, using the PCR-RFLP method and DNA sequencing. Seven wx microsatellite alleles, (CT)n repeats, (n = 9, 10, 11, 12, 16, 17 and 18) were found in 16 natural populations. Among them, the (CT)n repeats n = 9, 10, 11, 12 and 16 had the sequence G at the donor splice site and were associated with intermediate to high amylose wild rice strains. The (CT)18 allele, carrying the sequence T at this position, [(CT)18/T], was found only in wild rice strains with low amylose content. The (CT)17 allele was polymorphic for the G-T mutation at the donor splice site of intron 1 of the wx locus. Rice strains containing the (CT)17 allele with G at the donor splice site, [(CT)17/G], showed intermediate to high amylose content, while the allele with the sequence T at this position, [(CT)17/T], was found in rice strains with low amylose content. Variation of DNA-based genetic marker known as the waxy microsatellite corresponds to the amylose class of wild rice is important, as it should be useful in breeding programs designed to produce rice of desired grain quality and used as DNA marker in gene flow studies between cultivated and wild rice.