Abstract: A large number of studies have found that the five genes of LDHA, F-KER, DRD4, MSTN and CRY1 in the racing pigeons had single nucleotide polymorphisms, which were associated with the homing ability, making them become the potential SNP markers for molecular assisted breeding. The current detection methods were still based on the traditional RFLP, and the application of Sanger sequencing has not been reported. A detection method based on Sanger sequencing technology was established to identify the polymorphic sites in these five genes. The results showed that: the specific primers were designed for LDHA, F-KER, DRD4, MSTN and CRY1 genes in the racing pigeons to amplify the single nucleotide polymorphism sites. The obtained PCR products had single bands, showing good specificity. Further, the amplification and Sanger sequencing were performed on the feather DNA samples from 60 racing pigeons by using these primers. And the polymorphic genotyping results for each gene locus were successfully obtained. Finally, the distribution frequency of various genotypes among these 60 racing pigeons was statistically analyzed, and it was found that the frequencies of the relevant rare genotypes were all less than 5%, which were consistent with the frequencies reported in the foreign racing pigeon populations. The study showed that the established Sanger sequencing analysis method for SNP genotyping of racing pigeon performance-related genes had important application value for the breeding of racing pigeons with excellent genotypes.