Background Seasonal human influenza viruses can escape from antibody-mediated neutralization when amino acid changes occur in the hemagglutinin protein. Routine surveillance identified circulation of an A(H3N2) virus variant in the Netherlands with amino acid substitutions at hemagglutinin positions 158 and 189. These amino acid positions were previously responsible for antigenic change of influenza A(H3N2) viruses and potentially lead to escape of this variant from vaccine-mediated immunity. Aim To characterize the emergence and antigenic properties of N158K and K189R double substitution virus variants. Methods We analyzed the geographical and temporal dynamics of the double-substitution variant using a phylogeographic approach and used hemagglutination inhibition assays and antigenic cartography methods to map its antigenic properties. Results A(H3N2) viruses carrying K189R were first detected in Guatemala in June 2024, before subsequently gaining the N158K substitution, which was initially detected in Colombia in November 2024, followed by detection in the Netherlands in December 2024. However, detections within Europe remained almost entirely confined to the Netherlands. The proportion of viruses carrying the N158K and K189R substitutions increased to 16% - 24% per collection week of sequenced Dutch viruses during the peak of the epidemic of the 2024-2025 respiratory season. Antigenic characterization of viruses with N158K and K189R substitutions indicated that these are antigenically distinct from the A(H3N2) components of 2025-2026 Northern Hemisphere vaccines, showing 8-192-fold reduction in hemagglutination inhibition titers with antisera against the vaccine strain compared to antisera against the homologous virus. Conclusions Influenza A(H3N2) viruses with N158K and K189R escaped recognition by antibodies raised against the 2024-2025 and 2025/2026 Northern Hemisphere vaccine strains in hemagglutination inhibition assays. These variants circulated widely in the Netherlands during the 2024-2025 influenza season, raising concerns about reduced vaccine-mediated protection if such variants would spread more broadly during 2025-2026 Northern Hemisphere season.