Floret-opening time (FOT) refers to the time between florets opening and closing within a day, and is a crucial agricultural trait for reproductive development, thermal tolerance and hybrid breeding in rice. However, little is known about the genetic basis and genes controlling FOT in rice. Here, we report the genetic mapping and transcriptome analysis of FOT in the japonica rice cultivar G23. Combining the QTLseqr and GradedPool-seq (GPS) methods, we located a major quantitative trait locus (QTL), qFOT6, on chromosome 6 in multiple years and under different environments. Integrating RNA-seq analysis, we selected 13 potential candidate genes in the qFOT6 interval that might be associated with FOT in G23. Taken together, our work uncovers a major QTL and potential candidate genes for FOT in rice, thus providing invaluable clues for rice breeding.