In this study, 254 rice 3K germplasm resources were used as research materials, and they were planted in Changsha, Hunan Province, in 2021 and 2022, respectively. The panicle lengths of the various germplasm were measured at heading stage. Combined with germplasm genotypes for genome-wide association study, three panicle length QTL were detected on rice chromosomes 1, 5, and 6, named qPL-1, qPL-5, and qPL-6, respectively. These QTL explained phenotypic variation range from 9.06% to 28.27% of the variation of spike length. The candidate genes of qPL-1, qPL-5 and qPL-6 were finally obtained by combining the gene function annotation in the QTL interval and the significant difference of the panicle length of different haplotypes. With the co-localization of qPL-6 and sped1-D, Os01g0715600 and Os05g0132700 were identified as the new candidate genes for the regulation of spike length. Polymerization analysis of the elite haplotypes of Os01g0715600，Os05g0132700 and Os06g0597500 revealed a superposition effect within the three genes.