In poultry, Avian Leukosis Virus (ALV) causes high mortality and significant eradication costs, with no effective vaccines, hindering industry development. Improving resistance to ALV-J offers both economic and ecological value. The Qingyuan Partridge Chickens is a high-quality, possesses certain disease resistance capabilities. This study explored resistance molecular markers in Qingyuan Partridge Chickens through cell experiments, viral infection, and genomic resequencing. Results showed that interferon-alpha (IFN-α) expression significantly suppressed the ALV-J duplicated, with notable immune response differences between sexes. Genome-wide association study (GWAS) identified 62 single nucleotide polymorphisms (SNPs) linked to immune responses, cloacal viral positivity, and tissue indices. Selection for the C allele at chromosome 13 positions 8654609-8654610, the G allele at chromosome 9 position 21398106, and the G-T-C haplotype at chromosome 3 positions 59308643-59309366, while selecting against the T and A alleles at chromosome 1 positions 114656880 and 69131730, will effectively enhance immune function and reduce viral susceptibility. Gene Ontology analysis highlighted enrichments in biological processes like glial cell differentiation, cellular components such as side of membrane, and molecular functions like hydrolase activity. Quantitative PCR showed significant expression differences across immune organs in five genes, including IFN-α, interferon β (IFN-β), dual specificity phosphatase 1 (DUSP1), sosondowah ankyrin repeat domain family member C (SOWAHC), and dual specificity phosphatase 10 (DUSP10). These findings provide a genetic basis for breeding ALV-J-resistant Qingyuan Partridge Chickens through marker-assisted selection.