Based on common PCR, nested–PCR technology was developed to specially detect single nucleotide polymorphism (SNP). In this study, one tube nested–PCR technique was adopted to explore SNPs in four structural genes of rice, there were Fgr, a rice aroma gene and Pi–ta, Pi9 and Pigm, three rice blast resistance genes. Primers with effectively marked at targeting SNP sites were designed and four primers were mixed in one tube to amplify DNA fragments harboring mutation sites. In addition, reaction conditions including primer concentration and PCR program were optimized to identify different SNP genotypes in one step. Therefore, the one tube nested–PCR provided a feasible and efficient strategy to analyze SNP genotypes.