Abstract: The amylose content (AC) of rice is a key determinant of the grain cooking and eating quality. The Waxy (Wx) gene, which encodes granule-bound starch synthase I (GBSSI), is the major gene controlling AC in rice. The A – C substitution in exon 6 (Ex6A/C) alters the amino acid codon from serine to tyrosine and is associated with intermediate-AC rice (Wx-in). In this paper, we reported a perfect functional marker for the SNP Ex6A/C of Wx-in based on polymerase chain reaction with confronting two-pair primers (PCR-CTPP) in a single-tube PCR assay. Specificity and applicability of the PCR assay were verified in a Chinese mini core collection (MCC) of Oryza sativa L. and a breeding population. We recommend the use of the simple, inexpensive assay for routine genotyping for intermediate AC in the breeding population or discrimination of Wx-in genotypes in rice germplasm.