Abstract: We identified a mutant with extra leaves at the nodes of the panicle from a T-DNA insertion population constructed in our lab, and morphologically named it as a flag leaf mutant (J4 mutant). Analysis revealed that the extra leaves were abnormally developed, with short leaf blades, shortened epidemical cell lengths, and decreased numbers of vascular bundles in the leaf blade. Using TAIL-PCR and inverse-PCR analysis, we cloned the T-DNA flanking sequence in this mutant and accurately located the inserted T-DNA onto chromosome 2. Gene expression analysis revealed that AK100376, a T-DNA flanking gene, was obviously downregulated in both the J4 mutant and similar phenotype mutant neck leaf 1. Thus, AK100376 might be a candidate gene related to the phenotype in the J4 mutant. Here, we identified a flag leaf mutant and determined the phenotype-related candidate gene through expression analysis of the T-DNA flanking genes.