The endemicity of H9N2 avian influenza viruses (AIVs), particularly the Y280 lineage, poses persistent challenges to the poultry industry due to the limitations of inactivated vaccines, such as interference by maternally derived antibodies (MDAs) and incomplete suppression of viral replication. This study evaluated the immunogenicity and protective efficacy of a novel recombinant turkey herpesvirus vaccine expressing the hemagglutinin gene of H9N2/Y280 (rHVT-H9/Y280) in commercial Hy-Line Brown layers with high-MDA backgrounds. In a comparative challenge study, the rHVT-H9/Y280 vaccine induced complete protection against a homologous Y280 strain challenge at 4 weeks of age, whereas commercial inactivated vaccines failed to completely block replication, showing virus isolation rates of 16.7–25%. Long-term serological monitoring demonstrated that the rHVT-H9/Y280 vaccine elicited a robust humoral response characterized by persistent maintenance of high HI titers (>8.0 log₂) up to 39 weeks post-vaccination. These findings confirm that rHVT-H9/Y280 effectively overcomes MDA interference and provides protection by inhibition of viral replication in layer chickens, making it a promising candidate for the effective control of H9N2 AIV in endemic regions.

H9N2 low-pathogenic avian influenza remains endemic in Korean poultry production, where commercial broilers rely largely on maternally derived antibodies (MDA) transferred from vaccinated breeder hens. This study quantified MDA transfer and decay and evaluated protection against homologous Y280-lineage H9N2 challenge. Paired sera were collected from breeder hens and day-old progeny on six commercial farms. MDA decay was monitored in 90 broilers from 1 to 22 days of age, and 32 birds with individual HI titers of 0, 2, 4, or 6 log₂ were challenged intranasally at 7 days of age. Transfer efficiency averaged 18.2 ± 11.5%, and MDA had an estimated half-life of 3.2 days. Clinical signs occurred in 8/8, 8/8, 7/8, and 0/8 birds in the MDA 0, 2, 4, and 6 groups, respectively. Cecal tonsil virus detection decreased from 87.5% in the MDA 0 group to 37.5% in the MDA 6 group, although the pairwise difference was not statistically significant (p = 0.119). Oropharyngeal shedding occurred in all challenged birds at comparable titers. These findings suggest that higher MDA levels are associated with reduced clinical disease and lower cecal tonsil virus detection but do not prevent oropharyngeal shedding under the conditions tested.