Control of the amylose content is a major strategy for breeding rice with improved eating quality. To identify the low-amylose gene of the low-amylose rice cultivar Oborozuki, quantitative trait loci (QTL) and sequence analyses were conducted on 90 progeny obtained by crossing the two japonica cultivars Hokka287 (a donor parent of Oborozuki) and Hokkai-PL9. One QTL for low amylose content was detected on the WX1 locus on the short arm of chromosome 6. This gene was designated Wx1-1 for the Hokkai287 allele, and is a novel allelic gene for the WX1 locus with a 37-bp deletion in intron 10. Homozygote genotype for Wx1-1 decreased the amylose content by 7.8%. Primer sets Wx-U1L3 designed to amplify the genome region containing the deletion clearly differentiated cultivars containing Wx1-1 from others. This DNA marker is useful for breeding cultivars with low amylose contents. Another QTL, qAC9.3, was detected on the short arm of chromosome 9. Hokkai-PL9-homozygote allele for qAC9.3 decreased the amylose content by 2.6%. QTL analyses showed that Wx1-1 and the Hokkai-PL9 allele of qAC9.3 have an additive effect in decreasing the amylose content. The two genes are useful tools in marker-assisted selection when breeding rice with a controlled amylose content for improved eating quality.