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Background Surveillance of influenza virus circulation is crucial for monitoring the seasonal epidemiological dynamics of the disease. At the Regional Reference Laboratory of the Hygiene Unit of San Martino Hospital in Genoa, Italy, molecular diagnostics and influenza virus characterization are routinely performed.During the 2024–2025 influenza season, co-circulation of all three viral strains was observed in comparable proportions, whereas in the current season only influenza A viruses have been detected showing a significant epidemiological shift. An in-depth molecular investigation was implemented to more accurately characterize and evaluate the detected cases.Materials and Methods During the 2025 - 2026 influenza season, a total of 4068 nasopharyngeal swab specimens were collected from patients presented to the hospital with influenza-like illness (ILI) and analyzed using a routine diagnostic multiplex real-time RT-PCR assay (Allplex Respiratory Panel 1-2-3, Seegene, South Korea), which enables influenza virus detection and subtyping.Twenty-six samples with suitable cycle threshold (Ct) values for next-generation sequencing (NGS) analysis were randomly selected. Library preparation was performed using the Illumina Microbial Amplicon Prep-Influenza A/B kit (Illumina), and sequencing was carried out on the iSeq 100 platform (Illumina). Raw sequencing data (FASTQ files) were processed using the DRAGEN Microbial Amplicon pipeline (Illumina). Consensus sequences were subsequently evaluated using Nextclade.Results Of 4068 specimens analyzed, 1354 (33,3%) were positive for respiratory viruses multiplex real-time PCR and among these 443 (32,7%) were positive for influenza virus infection. Specifically, 214/443 (47,98%) samples tested positive for Influenza A/H3N2 and 229/443 (52,02%) for Influenza A/H1N1. No Influenza B viruses were detected ( figure 1).The remaining 902 samples that tested positive for other respiratory viruses exhibited a heterogeneous infection pattern, as illustrated in figure 2.Nextclade analysis revealed that H3N2 viruses belonged almost exclusively to clade K (with one J.2.4 variant), while all H1N1 viruses were assigned to clade D.3.1.1.Conclusions The 2025-2026 season marks a major shift in viral influenza virus circulation, dominated by Influenza A and the exclusion of Type B. The dominance of clades K and D.3.1.1 suggests an ongoing antigenic drift that may impact vaccine effectiveness and clinical severity. These findings underscore the importance of genomic surveillance in predicting seasonal trends and optimizing antiviral strategies.Abstract SC8 Figure 1–2