The flag leaf-related trait is important for the rice yield. The genetic mechanism of flag leaf length in rice was analyzed to lay a foundation in breeding of new varieties with ideal plant types and improve yield performance. In this study, the F2 population was constructed, using and Balilla (Oryza saliva L. sub sp. japonica; short flag leaves; female) pollinated with the O. longistaminata (long flag leaves; male). Based on the data of phenotype and genotype of the F2 populations in three years, the QTL of flag leaf length and their genetic effects were analyzed. Ten QTL modulating flag leaf length were detected on chromosome 3, 4, 5, 7, 8, 9, 11 and 12 of rice. Among them, the QTL qFLL9-1 was identified as the major locus, while the other two QTL qFLL4-1 and qFLL11-1 were newly identified. We further generated a high generation backcross population BC6F2 targeting qFLL9-1, and demonstrated this locus with an important role in the population. These results will benefit for future fine mapping of this QTL modulating flag leaf length, and the development and utilization of Oryza longistaminata.