Influenza 2024–2025 season in France was characterized by prolonged duration, unusual co-circulation of all three viruses (A(H1N1)pdm09, A(H3N2), B/Victoria) with several subclades, and substantial healthcare impact. We aimed to investigate the impact of influenza genetic diversity on vaccine effectiveness (VE). A test-negative design study was conducted to estimate VE in a large cohort from the RELAB network of community-based laboratories (n = 77,704 patients). A subset of sequenced samples (n = 2,119 patients) allowed VE estimation for several clades and subclades as well as comparison of subclade distribution by vaccination status. Vaccine coverage was 45% in patients aged 65 years and older (65+). VE based on PCR-confirmed infections was 44% (95% CI: 41-48%) and lower in 65+ individuals, at 25% (95% CI:18–31%) especially for type A virus (23%; 95% CI: 13-32%) compared to type B virus (57%; 95% CI: 35-72%). Sequencing-confirmed VE among individuals vaccinated 15 days to 3 months prior testing, was 41% (95% CI: 14–60%) for A(H1N1)pdm09 and 47% (95% CI: 21–64%) for its main subclade 5a.2a(C.1.9.3); A(H3N2) estimate was 30% (95% CI:5–48%) and 31% (95% CI:4–50%) for 2a.3a.1(J.2) sublclade. The emerging A(H1N1)pdm09 5a.2a.1 (D.3.1) subclade was significantly more frequent among vaccinated individuals compared to unvaccinated. The low vaccine coverage combined with the notably low effectiveness against A(H3N2) and for type A in elderly may have contributed to the high influenza activity this season. The emergence of A(H1N1)pdm09 5a.2a.1 (D.3.1) raises uncertainty and requires surveillance.