Because the rice varieties with resistance genes are easy to be overcome by pathogens, it is necessary to further explore new rice blast resistance genes. We used 470 germplasm accessions of the Rice Diversity Group II (RDP-II) to evaluate their disease resistance at the rice seedling stage in a hotspot blast nursery (Taojiang, Hunan, China) and identified rice blast resistance loci using genome wide association study (GWAS). Twenty-five varieties with high levels of resistance at the seedling stage were identified which can be used as resistant breeding materials. The disease phenotypes and 700 000 SNP genotypes were used to perform GWAS with the MLM method. A total of 24 resistance-associated loci were identified in the rice genome, which were distributed on 10 chromosomes except chromosome 4 and 7. Among these loci, 5 loci were overlapped with 6 cloned or mapped genes related to rice blast resistance, and the remaining 19 loci were new loci. In addition, the analysis of the rice subpopulations showed that the average resistance level of tropical japonica subpopulation was the highest, and the lowest was the temperate japonica subpopulation. These findings provided DNA markers for molecular-assisted breeding of blast resistance and genomic location information for subsequent cloning of these genes.