In this study, we constructed an F₂ isolated population derived from a cross between a dwarf wild rice mutant with shortgrain and a cultivar variety KJ01 with longgrain. And the genetic analysis for grain length of the F₂ population was performed. By using 132 pairs of polymorphic molecular markers evenly distributed on 12 chromosomes of rice, we conducted QTL mapping and major QTLs analysis, which would establish a foundation of further cloning the novel major grain length gene and provide a theoretical basis for grain shape breeding of rice. The results were as follows: (1) grain length trait of the isolated population F₂ was quantitative trait controlled by multiple genes. (2) By QTL linkage analysis of 543 F₂ individuals, a linkage map of 1 713.94 cM controlling rice grain length was constructed, and a total of 24 QTLs were detected, and only 3 QTLs showed additive genetic effects, the others showed negative genetic effects. (3) The three major QTLs located in interval markers of PSM379-RID24455, RID24455-RM15689, and RM571-RM16238 on chromosome 3, which explained phenotypic variation for 54.85%, 31.02%, and 7.62, respectively. (4) The grain length QTL located in interval markers of PSM379-RID24455 was the major QTL newly discovered in this study.