H9N2 avian influenza viruses (AIVs), which are enzootic in poultry and possess spillover potential to humans, represent a considerable global public health threat. Since 2021, we have observed an unprecedented decline in the ability of H9N2 viruses to agglutinate chicken red blood cells (CRBCs), a change that impairs effective detection in routine surveillance. Substituting CRBCs with turkey or guinea pig erythrocytes in hemagglutination assays effectively restored virus detectability. Through integrated bioinformatic and biological approaches, we identified that this phenotype is associated with specific amino acid substitutions at residues 131 and 132 of the 130-loop in the hemagglutinin (HA) protein. The HA motif 131/132-NT emerged in 2015 and rapidly increased in frequency, becoming the dominant pattern since 2021 in chickens and humans. Critically, the 131/132-NT motif attenuated viral binding affinity to short-chain α2,6-linked sialic acid receptors, explaining the loss of agglutination with CRBCs. Animal experiments further demonstrated that viruses carrying the HA 131/132-NT mutations maintained strong infectivity in chickens but altered tissue tropism in mice, showing reduced replication in the lungs. Collectively, our findings reveal a potential surveillance gap caused by reduced hemagglutination activity of H9N2 viruses, which may compromise the sensitivity of CRBC-based detection and potentially lead to under-detection of circulating viruses. Incorporating turkey or guinea pig red blood cells into surveillance protocol is therefore recommended to enhance detection sensitivity.