Objective To explore the effect of initiation codon mutation within tva receptor gene (tva c.3G>A) on resistance of chickens to infection by avian leukemia virus subgroup A (ALV-A).Method Sanger sequencing and RT-PCR were used to verify the presence of tva c.3G>A mutation in Chinese yellow-feathered broilers. The effect of tva c.3G>A mutation on infection of chickens by RCASBP(A)-GFP fluorescence reporter virus in vitro was detected using flow cytometry. The effect of tva c.3G>A mutation on infection of chickens by ALV-A was investigated using ALV-A challenge test in vivo. Direct sequencing was used to genotype tva c.3G>A mutation site within Chinese yellow-feathered broiler lines.Result The results of Sanger sequencing and RT-PCR identified the mutation from G to A on the third base in the coding region of tva gene of Chinese yellow-feathered broilers, which caused the mutation from ATG to ATA in the initial codon sequence of tva gene. The result of flow cytometry showed that chicken embryo fibroblasts (CEFs) of wild-type tva c.3G/G were susceptible to infection by RCASBP(A)-GFP, while the homozygous mutant tva c.3A/A CEFs were resistant to infection by RCASBP(A)-GFP, indicating that tva c.3G>A mutation led to chicken resistance to infection by RCASBP(A)-EGFP in vitro. The results of ALV-A challenge test in vivo also indicated that tva c.3G>A mutation led to chicken resistance to infection by ALV-A. Genotyping of tva c.3G>A revealed that homozygous resistance genotype tva c.3A/A was present in lines CB01, CB08, CB10 and CB15, with the frequencies of 0.10, 0.15, 0.23 and 0.08, respectively.Conclusion The tva c.3G>A mutation causes chicken resistance to infection by ALV-A in vitro and in vivo, and the tva c.3G>A mutation site can be used as the genetic resistance site of ALV-A.