The pubescence of the leaf blade surface is an important agronomic characteristic for rice morphology and significantly influences rice growth as well as physiological characteristics. This characteristic was analyzed in F1 and F2 plants derived by crossing cultivar 75-1-127 with the indica cultivar Minghui 63, as well as the glabrous cultivar Lemont and indica cultivar 9311. Results indicated that the pubescence of the leaf blade surface was a dominant trait and controlled by a single gene. The GL6 gene was primarily mapped on rice chromosome 6 with recessive F2 population derived from 75-1-127/Minghui 63 by combining bulked segregation analysis and recessive class analysis using the Mapmaker3.0/MapDraw software. The genetic distances between the simple sequence repeat markers RM20491 and RM20547 were 7.2 and 2.2 cM, respectively. The GL6 gene was fine mapped in the interval between InDel-106 and InDel-115 at genetic distances of 0.3 and 0.1 cM, respectively. The large, recessive F2 population was derived from 75-1-127/Minghui 63. A high-resolution genetic and physical map of GL6 was constructed. Derived from the map-based sequences published by the International Rice Genome Sequencing Project, the GL6 gene was localized at an interval of 79 (japonica) and 116.82 kb (9311) bracketed by InDel-106 and InDel-115 within the BAC accession numbers AP008403 and AP005760. Seven annotated genes (japonica) and eight annotated genes (9311) were present. The basis was further set for GL6 cloning and function analysis.