Objective To establish a new SNP genotyping method using Pfu DNA polymerase extend phosphorothioate-modified primers in a single tube bi-directional allele specific amplification ( SB-ASA) ． Methods For rat dbSNP loci rs8149053， we designed two outer and two inner allele-specific primers that were 3 ’-terminal phosphorothioate modified． Pfu DNA polymerase was used to specifically amplify the genome template and the SNP genotype was determined by different target segment DNA eletrophoresis． Results Different length segments represented different genotypes that were validated by sequencing． Conclusions Pfu polymerase genotyping assay is a rapid and specific method for detecting the genotype of the known SNP in a single PCR reaction．