Root biomass is an important determinant of seedling vigor that contributes to water and nutrient acquisition in rice (Oryza sativa L.). In this study, we evaluated the total root weight (TRW) of 127 Korean rice cultivars, comprising 110 temperate japonica (TEJ) and 17 Tongil-type cultivars, under hydroponic conditions. Population structure analysis separated the accessions into two major groups corresponding primarily to the TEJ and Tongil-type cultivars. A genome-wide association study (GWAS) was conducted using the FarmCPU, BLINK, and MLM approaches, all three of which detected a common association signal for TRW on chromosome 7. Further GWAS analysis using only the 110 TEJ cultivars revealed an association signal in the same genomic region, thereby providing evidence of a possible relationship between this region and the variation in TRW within the TEJ cultivars. Additionally, by performing linkage disequilibrium block analysis, we identified a candidate region spanning approximately 369 kb, containing 43 genes. On the basis of gene expression and haplotype analyses, Os07g0517000, Os07g0520400, and Os07g0520900 were selected as putative candidate genes. However, given that the observed haplotype differences were partially associated with the varietal-group composition, their effects should be interpreted with caution. These results will provide genomic information for gaining a better understanding of variations in seedling root biomass and contribute to further validation of genes associated with root development in rice.