Rice grain width and shape play a crucial role in determining grain quality and yield. The genetic basis of rice grain width was dissected into six additive quantitative trait loci (QTL) and 11 pairs of epistatic QTL using an F(7) recombinant inbred line (RIL) population derived from a single cross between Asominori (japonica) and IR24 (indica). QTL by environment interactions were evaluated in four environments. Chromosome segment substitution lines (CSSLs) harboring the six additive effect QTL were used to evaluate gene action across eight environments. A major, stable QTL, qGW-5, consistently decreased rice grain width in both the Asominori/IR24 RIL and CSSL populations with the genetic background Asominori. By investigating the distorted segregation of phenotypic values of rice grain width and genotypes of molecular markers in BC(4)F(2) and BC(4)F(3) populations, qGW-5 was dissected into a single recessive gene, gw-5, which controlled both grain width and length-width ratio. gw-5 was narrowed down to a 49.7-kb genomic region with high recombination frequencies on chromosome 5 using 6781 BC(4)F(2) individuals and 10 newly developed simple sequence repeat markers. Our results provide a basis for map-based cloning of the gw-5 gene and for marker-aided gene/QTL pyramiding in rice quality breeding.