Grain length in rice plays an important role in determining rice appearance, milling, cooking and eating quality. In this study, the genetic basis of grain length was dissected into six main-effect quantitative trait loci (QTLs) and twelve pairs of epistatic QTLs. The stability of these QTLs was evaluated in four environments using an F7 recombinant inbred line (RIL) population derived from the cross between a Japonica variety, Asominori, and an Indica variety, IR24. Moreover, chromosome segment substitution lines (CSSLs) harboring each of the six main-effect QTLs were used to evaluate gene action of QTLs across eight environments. A major QTL denoted as qGL-3a, was found to express stably not only in the isogenic background of Asominori but also in the recombinant background of Asominori and IR24 under multiple environments. The IR24 allele at qGL-3a has a positive effect on grain length. Based on the test of advanced backcross progenies, qGL-3a was dissected as a single Mendelian factor, i.e., long rice grain was controlled by a recessive gene gl-3. High-resolution genetic and physical maps were further constructed for fine mapping gl-3 by using 11 simple sequence repeat (SSR) markers designed using sequence information from seven BAC/PAC clones and a BC4F2 population consisting of 2,068 individuals. Consequently, the gl-3 gene was narrowed down to a candidate genomic region of 87.5 kb long defined by SSR markers RMw357 and RMw353 on chromosome 3, which provides a basis for map-based cloning of this gene and for marker-aided QTL pyramiding in rice quality breeding.