Abstract: The rice Pik locus that contains multiple blast resistance genes, such as Pi-k, Pi-km, Pi-kp, Pi1 and Pi-kh, has great utilization value in rice breeding for blast resistance in China. In the present study, we investigated the genotypes at the Pik locus in 229 rice cultivars and important breeding materials. Based on PCR amplification using K- and N-type specific primer sets, 80 accessions were identified harboring the K1K2 haplotypes. The 80 K1K2 accessions were further accessed for the presence of Pi-k, Pi-kp and Pi1 using allele-specific markers in combination with sequencing identification of allele-specific SNPs. Heidao and Jinghu B were identified harboring the Pi-k resistance allele, and Ruanmigu and Enhui 99-64 were identified harboring Pi-kp and Pi1, respectively. In addition, an elite indica restorer line Minhui 3301 was identified harboring the K1K2 haplotypes. Allele-specific marker analysis and sequencing data revealed that the sequence of Minhui 3301 at Pik locus differs from the sequences of Pi-k, Pi-km, Pi-kp, Pi1 and Pi-kh, suggesting that Minhui 3301 might possess a new allelic gene at the Pik locus. In summary, we validated the distribution of the K1K2 haplotypes at the Pik loucs in 229 rice accessions. Our results provide a wealthy of information for developing and deploying resistant cultivars to control rice blast disease.